Components

ABSTRACT

A method and device structure are provided which enable an archive sample to be collected and detached relative to a device within which a series of processes, such as PCR are being provided. A chamber structure and method of use are provided in which a controlled and precise volume is obtained by control of the relative resistance to flow through various channels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Utility Application which claims benefit of Ser.No. 61/151,111, filed Feb. 9, 2009 in the United States, and of Ser. No.61/151,107, filed Feb. 9, 2009 in the United States, and of Ser. No.61/151,117, filed Feb. 9, 2009 in the United States, and of Ser. No.61/151,104, filed Feb. 9, 2009 in the United States, and whichapplication(s) are incorporated herein by reference. A claim of priorityto all, to the extent appropriate is made.

BACKGROUND OF THE INVENTION

The invention concerns improvements in and relating to analysis,particularly, but not exclusively, in relation to biological samples.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a methodof providing a storable sample, the method including:

-   -   a) introducing a sample to a device; and    -   b) conveying at least a part of the sample to a receiving        location to provide a storable sample.

The method may include conveying a liquid to the receiving location. Themethod may include conveying a DNA containing material to the receivinglocation.

The method may provide a storable sample of a sample on which one ormore processes and/or reactions are performed. The storable sample maybe provided before one or more processes and/or reactions are performed.The storable sample may be provided after one or more processed and/orreactions are performed.

The method may provide a storable sample which is one part of thesample. One or more other parts of the sample may be used in one or moreprocesses and/or reactions. The sample may have one or more processesand/or reactions performed on it prior to taking the one part of thesample to provide the storable sample.

The one or more processes and/or one or more reactions may include oneor more of: cell lysis, mixing, a surface based reaction, washing,elution, selective separation of DNA from one or more other materials,application of a magnetic field, removal of a magnetic field and one ormore repeats thereof. The one or more processes and/or one or morereactions may include one or more of amplification, PCR, detection anddenaturation.

The at least a part of the sample may be conveyed using one or morecomponents of the device. The components may include one or morechannels and/or chambers and/or valves.

The method may include one or more of the following steps:

passing the sample through one or more channels and/or chambers to mixthe sample with one or more fluids and/or solids;

increasing the temperature of the sample and/or a mixture including thesample, preferably whilst in a chamber;

holding the sample and/or a mixture including the sample in a chamberfor a period of time;

passing the sample through one or more further channels and/or furtherchambers;

retaining at least a part of the sample in a chamber, preferably on asurface of one or more solids, preferably using a magnetic field;

washing at least another part of the sample from the chamber where theat least a part of the sample is retained;

eluting the retained part of the sample into a fluid.

The method may include transferring at least a part of the sample from areaction chamber to the receiving location. The method may includepassing the storable sample through the reaction chamber and then on tothe receiving location. The method may include passing the storablesample through an inlet into the reaction chamber and out through aseparate outlet from the reaction chamber. The reaction chamber may be aPCR reaction chamber. Preferably the at least a part of the sample maybe transferred prior to performing a reaction in the reaction chamber.The at least a part of the sample may be transferred during theperformance of a reaction in the reaction chamber. The at least a partof the sample may be transferred after performing a reaction in thereaction chamber.

The method may provide sample to a reaction chamber, with part of thesample progressing to the receiving location when the amount of samplein the reaction chamber exceeds a predetermined amount.

The method may include transferring at least a part of the sample to thereceiving location before that part of the sample reaches a reactionchamber, particularly a PCR reaction chamber. The method may includeproviding a split in a channel and/or chamber to feed part of the sampleto a reaction chamber and part of the sample to the receiving location,preferably without entering the reaction chamber. Preferably the atleast a part of the sample may be transferred prior to performing areaction in the reaction chamber. The at least a part of the sample maybe transferred during the performance of a reaction in the reactionchamber. The at least a part of the sample may be transferred afterperforming a reaction in the reaction chamber.

The method may provide sample to the reaction chamber, with part of thesample progressing to the receiving location when the reaction chamberis full of sample.

The at least a part of the sample transferred to the receiving locationmay be surplus sample.

The receiving location may be provided in a section of the device thatmay be detached from the device. The method may include detaching thereceiving location and/or section from the device, preferably after thestorable sample has been provided to the receiving location.

The method may include detaching the receiving location and/or sectionfrom the device by snapping the material joining the two. The method mayinclude detaching the receiving location and/or section from the deviceby breaking the material joining the two, for instance along a line ofweakness.

The method may include sealing the channel leading to the receivinglocation. The method may include sealing a channel and/or vent leadingfrom the receiving location. One or both of the seals are preferablyprovided on the section after the section is detached from the device.

The method may include sealing the channel on the device side of thelocation where the channel is detached when the section is detached fromthe device.

The method may seal the channel between the reaction chamber and thereceiving location in a horizontal section and/or a vertical sectionand/or diagonal section. Preferably the method seals the channel, at oneor more of the locations, on a horizontal section, and ideally betweenone or two vertical sections.

According to a second aspect of the invention there is provided adevice, the device having:

-   -   a) an entry location;    -   b) a channel connected to the entry location;    -   c) a receiving location, the receiving location being connected        to the channel.

The receiving location may be a container for a liquid. The receivinglocation may be a container for a DNA containing material.

The receiving location may be an integral part of the device.

The receiving location may be detachable from the device. The receivinglocation may be provided in a section of the device. Preferably thesection is detachable from the device. An area or line of weakness maybe provided between the device and the receiving location and/orsection. The section may be connected to the device at a line ofweakness.

The device may be provided with a first valve in the section, preferablyfor sealing the channel between the receiving location and the part ofthe channel disrupted when the section is detached from the device. Thedevice may be provided with a second valve not on the section,preferably for sealing the channel between the part of the channeldisrupted when the section is detached from the device and the remainderof the device. The device may be provided with a third valve in thesection, preferably for sealing the vent of the receiving locationand/or a channel leading from the receiving location to the vent of thereceiving location.

The receiving location may be a chamber. The receiving location may havean inlet in the top of the receiving location, the device having anorientation of use. The receiving location may have an outlet in the topof the receiving location, the device having an orientation of use.

The section may extend from the device. The section may be provided onone side of the device.

The maximum dimension of the section may be less than 20% the maximumdimension of the device, preferably less than 10%, more preferably lessthan 7.5% and ideally less than 5%. The maximum dimension of the sectionmay be the width of the section.

The device may have an orientation of use, the maximum height of thesection may be less than 20% the maximum height of the device,preferably less than 10%, more preferably less than 7.5% and ideallyless than 5%.

The device may have an orientation of use, the maximum width of thesection may be less than 20% the maximum dimension of the device,preferably less than 10%, more preferably less than 7.5% and ideallyless than 5%.

The device may have an orientation of use, the depth of the section maybe the same as the remainder of the device.

The volume of the section may be less than 5% of the volume of thedevice excluding the section, more preferably less than 3% and ideallyless than 1%.

The device may have an orientation of use, the channel between thedevice and the receiving location may include a horizontal sectionand/or a vertical section. Preferably the sealing of the channel, at oneor more of the locations, is provided on a horizontal section, andideally between one or two vertical sections.

The section may be provided with an identifier, such as a barcode. Theidentifier may be the same identifier information or include the sameidentifier information as an identifier provided on the remainder of thedevice.

The device may provide one or more processing locations and/or reactionlocations between the entry location and the receiving location. Thedevice may provide one or more processing locations and/or reactionlocations between the entry location and an output location. The devicemay provide a splitting location. The device may provide a splittinglocation from which a channel extends to the receiving location and/or aseparate channel extends to the output location. The channels may extenddirectly or via one or more intermediate chambers, locations or otherchannels. Preferably no processing locations and/or reaction locationsare provided between the splitting location and the receiving location.The device may provide one or more processing locations and/or reactionlocations between the splitting location and the output location.

The one or more processing locations may be channels and/or chambers.The processing locations may include one or more of: a mixing location,a washing location, a selective separation location for DNA from one ormore other material, an amplification process location, a location atwhich a magnetic field is applied and/or removed and/or varied and oneor more repeats of these.

The one or more reaction locations may be channels and/or chambers. Thereaction locations may be one or more of: a cell lysis location, asurface based reaction location, a selective separation location of DNAfrom one or more other materials, an amplification reaction location, alocation at which a magnetic field is applied and/or removed and/orvaried and one or more repeats of these.

The device may include one or more chambers. The device may include oneor more channels. The device may include one or more valves. The devicemay include one or more vents. The device may include one or more pumps,particularly electrochemical pumps.

The device may include a reaction chamber connected to the receivinglocation. The device may include an inlet to a reaction chamber and anoutlet from the reaction chamber to the receiving location. The reactionchamber may be a PCR reaction chamber.

The device may have an orientation of use, the device potentiallyincluding a reaction chamber, with an outlet positioned at apredetermined height in the reaction chamber. The reaction chamber mayhave a predetermined volume below the height of the outlet.

The splitting location may be provided with one channel connecting tothe receiving location and another channel connecting to a reactionchamber, particularly a PCR reaction chamber.

According to a third aspect of the invention we provide a method ofproducing a device, the method including:

-   -   a) forming an entry location in one or more components of the        device;    -   b) forming a channel in one or more components of the device;    -   c) providing a receiving location in one or more components of        the device;    -   d) assembling the one or more components to a device;    -   wherein the entry location is connected to the channel and the        channel is connected to the receiving location.

The first and/or second and/or third aspects of the invention mayinclude any of the features, options or possibilities set out elsewherein this application, including in the other aspects of the invention,the specific description of the embodiments and the drawings.

According to a fourth aspect, the invention provides a device, thedevice including one or more chambers.

The chamber may have an orientation of use. A chamber may be providedwith an inclined base. The base may be inclined at 20°+/−10°, preferably+/−5° and more preferably +/−3°.

The chamber may have a vertical side wall. Preferably the chamber hastwo side walls and both are vertical side walls. The side wall or sidewalls may be curved.

Preferably an inlet for a fluid and/or an inlet from a previous chamberis provided in the top wall of the chamber or in the top section of theside wall of the chamber. The top section may be the upper 20% of theheight of the chamber, more preferably upper 10%. Preferably the inletis provided in an upper corner of the chamber.

Preferably the outlet for the chamber is provided in the bottom wall ofthe chamber or in the bottom section of the side wall of the chamber.The bottom section may be the lower 10% of the height of the chamber,more preferably lower 5%. Preferably the outlet is provided in a lowercorner of the chamber.

Preferably the inlet and the outlet are provided in opposing corners ofthe chamber.

The chamber may provide a flow path for a liquid entering the chamber,that flow path being non-laminar. Preferably the flow path extends fromthe inlet down the inclined base of the chamber to an outlet.

The top wall, excluding any recesses present, may be may be horizontal+/−10°, preferably +/−5° and more preferably +/−3°.

Two or more such chambers may be provided in series.

The chamber may have an orientation of use. A chamber may be providedwith a horizontal base. The base may be horizontal +/−10°, preferably+/−5° and more preferably +/−3°.

The chamber may have an inclined side wall. Preferably the chamber hastwo side walls and both are inclined side walls. The side wall(s) may beinclined at between 50° and 85° to the horizontal, preferably between65° and 80°. The second side wall is preferably inclined in the oppositedirection to the first side wall. The first and second side wall may beinclined at the same angle.

Preferably an inlet for a displacing fluid and/or an inlet from a pumpis provided in the top wall of the chamber or in the top section of theside wall of the chamber. The top section may be the upper 20% of theheight of the chamber, more preferably upper 10%. Preferably the inletis provided in an upper corner of the chamber.

Preferably the outlet for the chamber is provided in the bottom wall ofthe chamber or in the bottom section of the side wall of the chamber.The bottom section may be the lower 10% of the height of the chamber,more preferably lower 5%. Preferably the outlet is provided in a lowercorner of the chamber.

Preferably the inlet and the outlet are provided in corners of thechamber on the same side of the chamber.

Preferably an inlet for a sample and/or an inlet from a samplecontaining chamber is provided in the bottom wall of the chamber or inthe bottom section of the side wall of the chamber. The bottom sectionmay be the lower 10% of the height of the chamber, more preferably lower5%. Preferably the inlet is provided away from the corners of the baseof the chamber.

The chamber may be provided with one or more vents. One or more of thevents may be provided with one or more valves, preferably valves movingfrom an open state to a closed state. One or more vents may be providedin the upper section of the chamber. The upper section may be the upper20% of the height of the chamber, more preferably upper 10%. One or morevents may connect to the chamber at a position higher than the inlet fora displacing and/or an inlet from a pump. The top wall, excluding anyrecesses present, may be may be horizontal +/−10°, preferably +/−5° andmore preferably +/−3°.

The chamber may have an orientation of use. A chamber may be providedwith a horizontal base. The horizontal base may provide a retentionlocation for one or more particles in the chamber. The one or moreparticles may be drawn to the retention location by a magnetic field.The highest strength magnetic field within the chamber is preferablyprovided at the base. The base may be horizontal +/−10°, preferably+/−5° and more preferably +/−3°.

The chamber may have an inclined side wall. Preferably the chamber hastwo side walls and both are inclined side walls. The side wall(s) may beinclined at between 20° and 80° to the horizontal, preferably between30° and 60°. The second side wall is preferably inclined in the oppositedirection to the first side wall. The first and second side wall may beinclined at the same angle.

Preferably an inlet for a wash and/or an inlet from a wash storagechamber is provided in the top wall of the chamber or in the top sectionof the side wall of the chamber. The top section may be the upper 20% ofthe height of the chamber, more preferably upper 10%. Preferably theinlet is provided in an upper corner of the chamber.

Preferably the outlet for the wash and/or outlet to a waste storagechamber is provided in the bottom wall of the chamber or in the bottomsection of the side wall of the chamber. The bottom section may be thelower 10% of the height of the chamber, more preferably lower 5%.Preferably the outlet is provided in a lower corner of the chamber.

Preferably the inlet and the outlet are provided in opposing corners ofthe chamber.

The chamber may provide a flow path for a liquid entering the chamber,that liquid being denser than the liquid in the chamber before.Preferably the flow path extends from the inlet down an inclined sidewall of chamber and/or across the bottom of the chamber to an outlet.Preferably the flow path passes through the region of the chamber withthe highest magnetic field strength.

Preferably an inlet for an eluent and/or an inlet from an eluent storagechamber is provided in the top wall of the chamber or in the top sectionof the side wall of the chamber. The top section may be the upper 20% ofthe height of the chamber, more preferably upper 10%. Preferably theinlet is provided in an upper corner of the chamber.

Preferably the outlet for the eluent and/or outlet to a further chamber,preferably a PCR reaction chamber, is provided in the bottom wall of thechamber or in the bottom section of the side wall of the chamber. Thebottom section may be the lower 10% of the height of the chamber, morepreferably lower 5%. Preferably the outlet is provided in a lower cornerof the chamber.

Preferably the inlet and the outlet are provided in opposing corners ofthe chamber.

The chamber may provide a flow path for a liquid entering the chamber,that liquid being denser than the liquid in the chamber before.Preferably the flow path extends from the inlet down an inclined sidewall of chamber and/or across the bottom of the chamber to an outlet.Preferably the flow path passes through the region of the chamber withthe highest magnetic field strength.

Preferably the inlet for the wash and/or inlet from a wash storagechamber is provided in one, preferably upper, corner of the chamber andan inlet for an eluent and/or an inlet from an eluent storage chamber isprovided in another, preferably upper, corner of the chamber. Preferablythe outlet for the wash and/or outlet to a waste storage chamber isprovided in one, preferably lower, corner of the chamber and an outletfor the eluent and/or outlet to a further chamber is provided inanother, preferably lower, corner of the chamber.

The chamber may be provided with one or more vents. One or more of thevents may be provided with one or more valves, preferably valves movingfrom an open state to a closed state. One or more vents may be providedin the upper section of the chamber. The upper section may be the upper20% of the height of the chamber, more preferably upper 10%. One or morevents may connect to the chamber at a position higher than the inlet fora wash and/or an inlet from a wash storage chamber and/or the inlet foran eluent and/or an inlet from an eluent storage chamber. One or morevents may be provided in a recess extending above the top wall of thechamber. The recess may be semi-circular. The top wall, excluding therecess if present, may be may be horizontal +/−10°, preferably +/−5° andmore preferably +/−3°.

The chamber may have an orientation of use. A chamber may be providedwith a curved base. The base may be semi circular. The base may be ahemisphere or proportion thereof. The chamber may be provided with acurved top. The top may be semi circular. The top may be hemisphericalor a portion thereof.

The top may be a larger volume than the bottom. The top hemisphere orportion thereof may be larger then the lower hemisphere or portionthereof

A transition surface may extend between the base of the chamber and thetop of the chamber.

The chamber may include a support location for one or more particles,such as a bead. The one or more particles may provide one or more or allthe reagents for a reaction, particularly an amplification, such as PCR.The support location may define a position of rest for the one or moreparticles. Preferably in the position of rest, the one or more particlesdo not block or obscure an inlet to and/or outlet from the chamber.Preferably in the position of rest at least 50%, preferably at least 60%and more preferably at least 70% of the surface area of the one or moreparticles are exposed to the chamber.

Preferably an inlet for a sample and/or an inlet from a previous chamberis provided in a side wall of the chamber. The inlet may be provided inthe mid section of the height of the chamber, preferably the middle 20%,more preferably the middle 10%.

Preferably the outlet for the sample and/or outlet to a receivinglocation and/or other chamber is provided in a side wall of the chamber.The outlet may be provided in the mid section of the height of thechamber, preferably the middle 20%, more preferably the middle 10%.

The inlet and the outlet are preferably provided opposite one another.The inlet and the outlet are preferably provided at the same height inthe chamber.

The chamber may have an orientation of use. A chamber may be providedwith a horizontal base and/or a horizontal top. The base and/or top, maybe horizontal +/−10°, preferably +/−5° and more preferably +/−3°.

The chamber may be provided with one or more side walls. The sidewall(s) may be vertical +/−10°, preferably +/−5° and more preferably+/−3°.

The chamber may include a support location for one or more particles,such as a bead. The one or more particles may provide one or more or allthe reagents for a reaction, particularly an amplification, such as PCR.The support location may define a position of rest for the one or moreparticles. Preferably in the position of rest, the one or more particlesdo not block or obscure an inlet to and/or outlet from the chamber.Preferably in the position of rest at least 50%, preferably at least 60%and more preferably at least 70% of the surface area of the one or moreparticles are exposed to the chamber.

Preferably an inlet for a sample and/or an inlet from a previous chamberis provided in the top of the chamber or in the upper section of thechamber. The upper section may be the upper 20%, more preferably theupper 10%.

Preferably the outlet for the sample and/or outlet to a receivinglocation and/or other chamber is provided in the top of the chamber. Theupper section may be the upper 20%, more preferably the upper 10%. Theinlet and the outlet may be the same.

Preferably the chamber is provided with a chamber filling outlet.Preferably fluid enters the chamber via the inlet and flows out of thechamber through the chamber filling outlet during the filling of thechamber. The chamber filling outlet is preferably provided in the baseor lower section of the chamber, for instance the lower 20% or morepreferably 10%.

The chamber may have an orientation of use. A chamber may be providedwith a horizontal base and/or a horizontal top. The base and/or top, maybe horizontal +/−10°, preferably +/−5° and more preferably +/−3°.

The chamber may be provided with one or more side walls. The sidewall(s) may be vertical +/−10°, preferably +/−5° and more preferably+/−3°.

The junction between the base and the side walls may be curved. Thejunction between the top and the side walls may be curved. The junctionbetween the top and the side walls may be provided by an intermediatewall. The intermediate wall may be inclined relative to the top and/orside walls.

The chamber may include a support location for one or more particles,such as a bead. The one or more particles may provide one or more or allthe reagents for a reaction, particularly an amplification, such as PCR.The support location may define a position of rest for the one or moreparticles. Preferably in the position of rest, the one or more particlesdo not block or obscure an inlet to and/or outlet from the chamber.Preferably in the position of rest at least 50%, preferably at least 60%and more preferably at least 70% of the surface area of the one or moreparticles are exposed to the chamber. The support location may beprovided by the base of the chamber.

Preferably an inlet for a sample and/or an inlet from a previous chamberis provided in the top of the chamber or in the upper section of thechamber. The upper section may be the upper 20%, more preferably theupper 10%.

The inlet may be provided in a corner of the chamber.

Preferably the outlet for the sample and/or outlet to a receivinglocation and/or other chamber is provided in the top of the chamber. Theupper section may be the upper 20%, more preferably the upper 10%. Theinlet and the outlet may be provided at the same height.

The outlet may be provided in a corner of the chamber.

An inlet channel may be provided which leads to the inlet. An outletchannel made be provided which leads away from the outlet. A by-passchannel may be provided for the chamber. The by pass channel may connecta part of the inlet channel to a part of the outlet channel.

The by-pass channel may be a continuation of the channel from which theinlet channel and/or outlet channel branch. The by-pass channel andchannel may have a common axis.

The by-pass channel may be a branch from the channel from which theinlet channel branches. The by-pass channel and/or inlet channel may beprovided with an axis which is not a continuation of the axis of thechannel from which they branch. Preferably, the by-pass channel isprovided with an axis which is not a continuation of the axis of thechannel from which it branches, with still more preferably the inletchannel being provided with on a common axis to that of the portion ofthe channel which adjoins it.

The by-pass channel may be a branch from the channel from which theoutlet channel branches. The by-pass channel and/or outlet channel maybe provided with an axis which is not a continuation of the axis of thechannel from which they branch. Preferably, the by-pass channel isprovided with an axis which is not a continuation of the axis of thechannel from which it branches, with still more preferably the outletchannel being provided with on a common axis to that of the portion ofthe channel which adjoins it.

Preferably one or more dimensions of the outlet channel are smaller thanthe corresponding dimension of the inlet channel. The value of the oneor more dimensions may be considered at the location within the inletchannel and/or outlet channel where that dimension has its lowest value.The one or more dimensions may include one or more or all of the widthand/or height and/or cross-sectional area. The cross-sectional area maybe measured perpendicular to the direction of flow in the inlet channeland/or outlet channel and/or perpendicular to the alignment or axis ofthe inlet channel and/or outlet channel.

The resistance to fluid flow provided by the outlet and/or outletchannel may be greater than the resistance to fluid flow provided by theinlet and/or inlet channel. The resistance to fluid flow provided by theoutlet and/or outlet channel may be greater than the resistance to fluidflow provided by the by-pass channel.

The path of least resistance for the fluid may be through the inlet andinto the chamber until the fluid reaches the outlet and/or outletchannel. The path of least resistance for the fluid may be through theby-pass channel once the fluid has reached the outlet and/or outletchannel.

The fluid flow may switch from the inlet channel to the by-pass channelwhen a predetermined volume of fluid is provided in the chamber.

The chamber may have an orientation of use. A chamber may be providedwith a curved base. The base may be semi circular. The base may be ahemisphere or proportion thereof. The chamber may be provided with a topwall, such as a planar top wall. The top wall may be provided in one ormore portions. The plane of one or more of those portions may bedifferent to the plane of one or more of the other portions. Preferablythe planes are parallel.

An inclined transition surface may extend between the base of thechamber and the side walls of the chamber. The side wall may connect tothe top of the chamber. The side walls may be vertical in theorientation of use.

The chamber may include a support location for one or more particles,such as a bead. The one or more particles may provide one or more or allthe reagents for a reaction, particularly an amplification, such as PCR.The support location may define a position of rest for the one or moreparticles. Preferably in the position of rest, the one or more particlesdo not block or obscure an inlet to and/or outlet from the chamber.Preferably in the position of rest at least 50%, preferably at least 60%and more preferably at least 70% of the surface area of the one or moreparticles are exposed to the chamber.

Preferably an inlet for a sample and/or an inlet from a previous chamberis provided in a side wall of the chamber. The inlet may be provided inthe lower section of the height of the chamber, preferably the lower30%, more preferably the lower 10%. Preferably the outlet for the sampleand/or outlet to a receiving location and/or other chamber is providedin a top wall of the chamber. The outlet may be provided in the topsection of the height of the chamber, preferably the top 20%, morepreferably the top 10%.

The inlet and the outlet are preferably provided opposite one another.The inlet and the outlet are preferably provided at different heights inthe chamber.

The chamber may at least in part be defined by a rotatable element. Therotatable element may provide one or more walls of the chamber. Therotatable element may provide the front, back and side wall of thechamber. The chamber may be a cylinder or section thereof. The rotatableelement may provide one or more of the front and back walls of thechamber, with the device providing the other walls not provided by thechamber. One or more through apertures may be provided in a wall orwalls of the chamber. The front and/or back walls may be planar.

One or more parts may be provided on the rotatable element and/or deviceto limit rotation of the rotatable element, for instance at the firstand/or second and/or third positions.

The rotatable element may be a snug fit within a recess in the device,such as a cartridge. One or more contacts between the rotatable elementand the device may be provided with a seal and/or sealing material.

The chamber may be rotated by engaging an actuator with the chamber, forinstance with the front or rear wall thereof.

The rotatable element may have a first position and a second position.In the first position one or more channels may be in fluid communicationwith the inside of the chamber. In the first position one or morechannels may not be in fluid communication with the inside of thechamber. In the second position one or more different channels may be influid communication with the inside of the chamber. In the secondposition one or more different channels may not be in fluidcommunication with the inside of the chamber.

In the first position an inlet channel may be in fluid communicationwith the inside of the chamber. In the first position an outlet channel,such as a venting channel, may be in fluid communication with the insideof the chamber. In the first position a further outlet channel, such asa discharge outlet channel, may not be in fluid communication with theinside of the chamber.

In the second position an inlet channel may not be in fluidcommunication with the inside of the chamber. In the second position anoutlet channel, such as a venting channel, may be in fluid communicationwith the inside of the chamber. In the second position a further outletchannel, such as a discharge outlet channel, may be in fluidcommunication with the inside of the chamber.

In the first position an inlet channel may be in fluid communicationwith the inside of the chamber. In the first position an outlet channel,such as a venting channel, may be in fluid communication with the insideof the chamber. In the first position a further inlet channel may not bein fluid communication with the inside of the chamber. In the firstposition a further outlet channel, such as a discharge outlet channel,may not be in fluid communication with the inside of the chamber.

In the second position an inlet channel may not be in fluidcommunication with the inside of the chamber. In the second position anoutlet channel, such as a venting channel, may not be in fluidcommunication with the inside of the chamber. In the second position afurther inlet channel may be in fluid communication with the inside ofthe chamber. In the second position a further outlet channel, such as adischarge outlet channel, may be in fluid communication with the insideof the chamber.

A third position may be provided. The third position may be intermediatethe first and second positions. In the third position the combination ofchannels in fluid communication with the chamber and/or not in fluidcommunication with the chamber may be different than in the first and/orsecond position. The third position may provide that no channels are influid communication with the inside of the chamber. One or more steps orprocesses may be applied to the contents of the chamber when in thethird position. The one or more steps or processes may include anamplification step and/or PCR step or one or more sub-steps thereof.

One or more of the channels may be used to inspect the contents of thechamber, for instance by introducing light and/or considering the lightreturning from the chamber.

According to a fifth aspect, the invention provides a method ofcontrolling the passage of one or more materials within a device, themethod including:

moving one or more materials from a channel into a chamber connected tothe channel;

moving one or more of the materials from the chamber into a channelconnected to the chamber.

According to a sixth aspect, the invention provides a method ofproducing a device, the method including:

forming a recess in one or more components of the device;

forming a channel in one or more components of the device;

assembling the one or more components to form a chamber from the recess;

wherein the chamber is connected to the channel.

The fourth and/or fifth and/or sixth aspects of the invention mayinclude any of the features, options or possibilities set out elsewherein this application, including in the other aspects of the invention,the specific description of the embodiments and the drawings.

According to a seventh aspect of the invention, there is provided adevice, the device having:

-   -   a) a chamber;    -   b) a channel; and    -   c) a vent element;    -   wherein the chamber is connected to the channel, the channel is        connected to the vent element and the vent element leads towards        the outside of the device.

The vent element may be an element which is separate from the channeland/or the channel walls. The vent element may be applied to thechannel, for instance to one or more of the channel walls.

The vent element may allow the passage of and/or be permeable to airand/or other gases.

The vent element may resist the passage of water and/or other liquids.The vent element may prevent the passage of and/or be impermeable towater and/or other liquids.

The vent element may resist the passage of particulate material. Thevent element may prevent the passage of and/or be impermeable toparticulate matter. The particulate material may be or include: cells,dust, DNA containing material.

The vent element may be hydrophobic. The vent element may be formed of ahydrophobic material. The vent element may include one or more surfacesprovided with a hydrophobic coating.

The vent material may be or include polypropylene. The vent material mayinclude a polysulphone based polymer coating.

The vent element may be or include a filter element.

The vent element may be provided in a vent chamber in the device. Thevent chamber may be filled by the vent element. The vent chamber mayhave an inlet from the channel and an outlet to the outside of thedevice. The outlet may lead directly to the outside of device or maylead via a vent channel to the outside of the device. The vent elementis preferably provided across the path between the inlet to the ventchamber and the outlet from the vent chamber. The vent chamber may havea circular cross-section, particularly perpendicular to the axis of thechannel and/or the vent channel. The vent chamber may be cylindrical.

The device may have an orientation of use. In the orientation of use,the vent element may be positioned above the channel. In the orientationof use, the vent element may be positioned above the chamber. In theorientation of use, the part of the channel which is connected to thevent element may be vertically orientated. In the orientation of use,the channel may include a further part which is horizontally orientated.

According to a eighth aspect of the invention, there is provided amethod of producing a device, the method including:

-   -   forming a recess in one or more components of the device;    -   forming a channel in one or more components of the device;    -   providing a vent element in one or more components of the        device;    -   assembling the one or more components to form a chamber from the        recess;    -   wherein the chamber is connected to the channel, the channel is        connected to the vent element and the vent element leads towards        the outside of the device.        According to an ninth aspect of the invention, there is provided        a method of controlling the passage of one or more materials        between the inside of a device and the outside of a device, the        method including:

moving one or more materials from a chamber into a channel connected tothe chamber;

moving one or more of the materials from the channel into a vent elementconnected to the channel;

moving one or more of the materials from the vent element to the outsideof the device.

The fluid pressure on the inside of the vent element may be greater thanthe fluid pressure on the outside of the vent. Preferably the fluidpressure on the inside of the vent element may be greater than the fluidpressure on the outside of the vent when a connection exists between theoutside of the device and channel and/or the chamber. Preferably thevent element is under positive pressure from the inside when aconnection exists between the outside of the device and the channeland/or the chamber. Preferably any flow of fluid through the ventelement is from the inside of the device to the outside of the device.

The method may include a first stage during which the fluid in thechannel is at a higher pressure than the pressure on the outside of thevent element. The method may include a first stage in which fluid flowsthrough the channel and flows through the vent element. Preferably thefluid of the first stage is a gas. Preferably the fluid of the firststage is air.

The method may include a second stage during which the fluid in thechannel is at a higher pressure than the pressure on the outside of thevent element. The method may include a second stage in which the fluiddoes not flow through the vent element. Preferably the fluid of thesecond stage is a liquid. Preferably the fluid of the second stage iswater.

The transition from the first stage to the second stage may occur whenthe boundary between a first fluid and a second fluid reaches the ventelement. The transition from the first stage to the second stage mayoccur when the boundary between a first fluid and a second fluid reachesa hydrophobic material. The boundary may be between a gas as the firstfluid and a liquid as the second fluid. The boundary may be between airas the first fluid and water as the second fluid. The method may includea second stage in which fluid flows through the channel

The seventh and/or eighth and/or ninth aspects of the invention mayinclude any of the features, options or possibilities set out elsewherein this application, including in the other aspects of the invention,the specific description of the embodiments and the drawings.

According to a tenth aspect, there is provided a device, the deviceincluding a valve.

Preferably the device only has two types of valve. Preferably all of thevalves of each of the two types are identical.

The valve may be an open to closed valve, preferably such that thechannel the valve is connected to, is open before the valve is activatedand is closed after the valve is activated. Preferably all the valves ofthe open to closed type are identical in terms of component parts and/orvolume and/or length and/or height and/or depth and/or meltable materialand/or orientation.

The open to closed valve may include a conduit which connects the valveto the channel to be acted on. The conduit may also connect to a valvereservoir, for instance provided with a meltable material, for instanceparaffin wax. The valve reservoir may be connected to a further conduit,such as a gas passage. The further conduit may be connected to a furthervalve reservoir, for instance provided with air.

Preferably the device has an orientation of use, in the orientation ofuse, the valve being provided above the channel the valve is to actupon. The section of the channel that the valve is to act upon may behorizontal, for instance +/−10°, preferably +/−5° and more preferably+/−3°. The conduit and/or further conduit may be vertical, for instance+/−10°, preferably +/−5° and more preferably +/−3°.

A heater may be provided for the valve. The heater may be providedoutside of the device, for instance on another component. The heater maydirectly or indirectly abut a part of the valve.

The transition from the open state to the closed state may be providedby applying heat to the valve. The heat may cause the meltable materialto become a liquid. The heat may cause the contents, particularly air,in the further valve reservoir to expand. Expansion of the contents ofthe further valve reservoir may assist in moving the contents of thevalve reservoir into the channel. The transition from open state to theclosed state may be provided by removing a heat source after a periodduring which heat was applied. The removal of the heat source may causethe meltable material to solidify in the channel.

The section of the channel the valve is to act upon may be providedbetween one or more further sections. One or more of the furthersections may be inclined from the horizontal, for instance by more than45°, preferably by more than 65° and more preferably by more than 80°.One, preferably two, of the further sections are preferably inclinedupwards relative to the section. One, preferably two, of the furthersections are provided adjacent the section and/or connected directlythereto.

One or more different melting point meltable materials may be used in adevice and/or within a single valve. Within a single valve, thedifferent melting point meltable materials may be used within a singlevalve reservoir or in separate valve reservoirs. The different meltingpoint meltable materials may be mixed with one another, for instancebefore and/or during and/or after activation of the valve. A lowermelting point material and a higher melting point material may beprovided. The higher melting point material may be provided with amelting point greater than 90° C., more preferably greater than 95° C.

The valve may be a closed to open valve, preferably such that thechannel the valve is connected to, is closed before the valve isactivated and is open after the valve is activated. Preferably all thevalves of the closed to open type are identical in terms of componentparts and/or volume and/or length and/or height and/or depth and/ormeltable material and/or orientation.

The closed to open valve may include a valve chamber which is a part ofthe channel, having an inlet from the channel and an outlet to thechannel. The valve chamber may include a meltable element, the meltableelement blocking the channel through the valve chamber in the closedstate. The meltable material may be paraffin wax. The valve chamber mayinclude a lower chamber section, preferably provided below the channeland/or flow path through the valve chamber.

Preferably the device has an orientation of use, in the orientation ofuse, the valve chamber being provided in a horizontal section of thechannel the valve is to act upon. The section of the channel that thevalve is to act upon may be horizontal, for instance +/−10°, preferably+/−5° and more preferably +/−3°.

A heater may be provided for the valve. The heater may be providedoutside of the device, for instance on another component. The heater maydirectly or indirectly abut a part of the valve.

The transition from the closed state to the open state may be providedby applying heat to the valve. The heat may cause the meltable materialto become a liquid. The heat may cause the meltable material to flowfrom the blocking position into the lower chamber section. The flow ofthe meltable material into the lower chamber section may open thechannel and/or flow path through the valve chamber. Pressure may beapplied behind the meltable material to assist its flow. The transitionfrom closed state to the open state may be provided by removing a heatsource after a period during which heat was applied. The removal of theheat source may cause the meltable material to solidify in the lowerchamber section.

According to a eleventh aspect, there is provide a method of producing adevice, the method including:

forming a channel in one or more components of the device;

forming a valve connected to the channel;

assembling the one or more components to form a device.

According to a twelfth aspect, the invention provides a method ofcontrolling the passage of one or more materials within a device, themethod including:

moving one or more materials from a first location in the device to asecond location in the device;

controlling the passage of the one or more materials using a valve.

The tenth and/or eleventh and/or twelfth aspects of the invention mayinclude any of the features, options or possibilities set out elsewherein this application, including in the other aspects of the invention,the specific description of the embodiments and the drawings.

Any of the aspects of the invention may include any of the followingoptions, features or possibilities.

The sample may be received from one or more of: a swab, a buccal swab, acotton swab, a soft swab, a solution, a suspension, an item of clothing,an item placed in the mouth, a cigarette or piece thereof, chewing gumor saliva.

The sample may be a skin sample, blood sample, cell sample, bodily fluidsample, hair sample, saliva sample or sample containing one or more ofthese.

The sample may be a forensic sample. The sample may be a medical sample.

The analysis may be for diagnostic purposes. The analysis may be forforensic purposes.

The analysis may be for use in the consideration of marker targets,diagnostic assays, disease markers, biobanking applications, STR basedtargets in transplants, identification of drug resistant microorganisms,blood testing, mutation detection, DNA sequencing, food analysis,pharmogenetics and pharmogenomics, medical fields, biotech fields, indetermining familial relationships, paternity testing and pedigreetesting in animals.

The analysis may be for use in border control, security or customssituations and/or uses.

The device may be a microfluidic device. The instrument may incorporatea microfluidic device. The device may be a device processing a sample ofless than 1 ml, possibly less than 500:1, possibly less than 250:1,potentially less than 200:1, possibly less than 175:1, possibly lessthan 50:1, preferably less than 30:1, more preferably less than 20:1,potentially less than 10:1 in one or more steps. The device may be adevice processing a fluid, particularly a liquid, of less than 50:1,preferably less than 30:1, more preferably less than 20:1, potentiallyless than 10:1 in one or more steps.

The device may process and/or contain a fluid, particularly a liquid, ofless than 50:1, preferably less than 30:1, more preferably less than20:1, potentially less than 10:1 in one or more of the following steps:a sample receiving step and/or sample preparation step and/or sampleextraction step and/or sample retention step and/or purification stepand/or washing step and/or elution step and/or amplification step and/orPCR step and/or denaturing step and/or investigation step and/orelectrophoresis step and/or analysis step and/or results output step.

The device may incorporate one or more channels or chambers with amaximum dimension of less than 1000:m, possible less than 750:m andpreferably less than 550:m. The device may incorporate one or morechannels or chambers with a maximum dimension of less than 500:m,possible less than 250:m and preferably less than 100:m.

The device may include a chambers provided with one or more reagents.One or more chambers may be so provided. The reagents may be different.The reagents may be in liquid form. The reagents may be provided onand/or in the surface of a solid. The reagents may be provided on and/orin the surface of a solid in gel form. The solid may be one or morebeads. The solid may be magnetic. The reagents may be released as aresult of a change in conditions. The change in conditions may be achange in temperature and/or a change in pH.

One or more reagents may be provided for cell lysis. One of morereagents may be provided for a selective extraction of DNA containingmaterial from other material. One or more reagents may be provided forwashing. One or more reagents may be provided for elution, particularlyfrom the surface of a solid. One or more reagents may be provided foramplification, particularly PCR based amplification. One or morereagents may be provided for denaturing. One or more reagents may beprovided for electrophoresis.

Preferably the device has a stored form and a use form. In the use form,the sample to be processed may be loaded into the device. Preferably oneor more reagents are pre-loaded into the device and/or are present inthe device when in the stored form. One or more reagents may be loadedinto the device in the use form.

The device and/or method may include one or more pumps. Preferably thedevice only includes pumps of a single type. Preferably the pumps of thesingle type are identical with respect to chamber shape and/or electrodepositions and/or electrode materials and/or orientation and/or chambervolume and/or pump electrolyte and/or pump electrolyte concentration.

One or more, preferably all, of the pumps may be electrochemical pumps.

The device may have an orientation of use, preferably one electrode inthe pump chamber is provided above the other. The pump chamber may havea height greater than its width. The pump chamber may have a widthgreater than its depth.

The pump chamber may have an outlet. Preferably the outlet is providedin the upper section of the pump chamber. The upper section may be theupper 20%, preferably 10%, and more preferably 5% of the height of thechamber. The outlet may be in the top wall of the chamber.

The pump chamber may contain NaCl. The molarity of the electrolyte inthe pump chamber may be between 0.2M and 3M, preferably 1M+/−15%.

The electrophoresis step and/or electrophoresis cartridge section may beprovided with a channel, for instance a capillary for electrophoresis.

The channel may be provided with a matrix. Preferably the matrix resiststhe passage of elements, the resistance being related to the size of theelement. Preferably different size elements migrate through the matrixat different rates, the larger migrating slower.

The channel may be provided with an inert bed of particulate material toform the matrix.

The channel may be provided with a gel, particularly a polymer gel. Thechannel may be provided with polyhydroacrylamide, polydimethylacrylamideor mixtures there of. The channel may be provided with a cross-linkedpolymer. The cross-linking of the polymer may be provided in situ.

One or more surfaces of the channel may be treated, for instance with ahydrophilic coating, for instance poly(hydroxyethlacrylamide).

The channel may be provided with a matrix during electrophoresis. Thechannel may be provided without a matrix prior to electrophoresis, withthe matrix being introduced before electrophoresis commences. The matrixor a material for forming the matrix may be stored at a location removedfrom the channel in which electrophoresis is provided. The matrix ormaterial for forming the matrix may be stored in a chamber. The chambermay be connected by a channel to the channel in which electrophoresis isprovided.

The matrix and/or material for forming the matrix may be altered beforeuse in the electrophoresis step. The alteration may be provided beforeand/or during and/or after the matrix and/or material for forming thematrix is provided in the channel. The alteration may be polymerisation.The alteration may be caused and/or triggered by heating and/or theapplication of light, such as UN light. The alteration may be applied toall of the matrix and/or material for forming the matrix or only a partthereof. One or more parts of the matrix may be prevented fromalteration, for instance by masking those parts and/or excluding heatand/or excluding light from them.

The sample receiving step may include the transfer of a sample fromoutside the device and/or instrument, to inside the device and/orinstrument. The sample receiving step may receive the sample from acollection device or from a storage device. The sample receiving stepmay include the transfer of the sample to a channel or chamber withinthe device.

The sample preparation step may include contacting the sample with oneor more reagents and/or one or more other components. The reagentsand/or other component may be used to prepare the sample for one or moreof the subsequent steps.

The sample extraction step may be part of or separate from the samplepreparation step. The sample extraction step may include contacting thesample with one or more reagents and/or components which select thesample component(s) relative to one or more waste components in thesample. The selected sample component(s) may be removed from the wastecomponent(s) and/or the waste component(s) may be removed from theselected sample components. The waste component(s) may flow away fromthe extraction step. The waste component(s) may be washed away from theextraction step using one or more further reagents and/or components.

The sample retention step may be a part of or may be separate from thesample preparation step and/or sample extraction step. The sampleretention step may include contacting the sample with one or morereagents and/or components which retain the sample component(s) relativeto one or more waste components in the sample. The sample component(s)may be retained on one or more beads. The beads may be magnetic. Theretained sample component(s) may be removed from the waste component(s)and/or the waste component(s) may be removed from the retained samplecomponents. The waste component(s) may flow away from the retentionstep. The waste component(s) may be washed away from the retention stepusing one or more further reagents and/or components. The wastecomponent(s) may flow past the location of retention. The wastecomponent(s) may be washed away using one or more further reagentsand/or components which flow past the location of retention.

The retained and/or selected sample may be eluted, preferably with theeluent conveying the retained and/or selected sample to the next step.

The purification step may be a part of or may be separate from thesample preparation step and/or sample extraction step and/or sampleretention step. The purification step may separate the selected samplecomponents, for instance DNA, from one or more waste components of thesample, for instance cellular material, PCR inhibitors and chemicalinhibitors.

The washing step may be a part of or may be separate from the samplepreparation step and/or sample extraction step and/or sample retentionstep and/or purification step. The washing step may remove one or morecomponents of the sample from the location of one or more othercomponents of the sample.

The elution step may be a part of or may be separate from the samplepreparation step and/or sample extraction step and/or sample retentionstep and/or purification step and/or washing step. The elution step mayremove one or more components of the sample from a first form into asecond form. The first form may be bound to a surface or substrate, forinstance on a bead. The second form may be in a liquid, for instance theeluent.

The amplification step may include contacting the sample with one ormore reagents and/or components to cause amplification. Theamplification step may include contacting the sample with conditions,preferably of a cyclic nature, to cause amplification. The amplificationmay be provided by a PCR step.

The denaturing step may prepare the sample for electrophoresis. Thedenaturing step may include contacting the sample with one or morereagents and/or components. The denaturing step may include contactingthe sample with conditions, preferably of a cyclic nature, to causedenaturing.

The investigation step may provide a characteristic for one component ofthe sample which differs from the characteristic for one or more othercomponents of the sample. The characteristic may be one or moredetectable positions and/or one or more signals and/or one or moreintensities and/or one or more colours and/or one or more concentrationsand/or presence of one or more characteristics and/or absence of one ormore characteristics.

The electrophoresis step may be part of or may be separate from theinvestigation step. The electrophoresis step may include transferringthe sample to a start location for electrophoresis and/or a mobilitybased separation and/or a size based separation. The start location maybe in a channel. The electrophoresis step may include one or morevoltage conditions. One or more voltage conditions may be used totransfer the sample to the start location. One or more voltageconditions may be used to provide the separation.

The analysis step may establish one or more of the characteristics ofthe sample. The analysis may interrogate the instrument, particularlythe device, and/or may seek a response from the instrument, particularlythe device. The analysis may subject the instrument, particularly thedevice, to an operation, for instance the application of light. Theanalysis may consider the response to the operation, for instance thelight returning.

The analysis step may include one or more operations involving aninteraction with the device. The analysis step may include one or moreoperations not involving an interaction with the device. One or more ofthe interactions may be electromagnetic interactions.

The analysis step may apply light to the device. The analysis step mayreceive light from the device. The analysis step may establish therelative position of the elements having a characteristic, for instancean allele having a fluorescent dye. The analysis step may establish therelative size of the elements having a characteristic, for instance anallele having a fluorescent dye. The analysis step may generate one ormore results. The light may be of visible and/or non-visiblewavelengths. The results output step may display the one or more resultsfrom the analysis step and/or a processed form thereof.

The results output step may transmit the one or more results from theanalysis step and/or a processed form thereof to a remote location. Theresults output step may compile the one or more results into atransmission form. The transmission may be via a telecommunicationsnetwork. The results may be provided in a format compatible with one ormore software applications.

The results output step may be followed by a further processing step.The further processing may interpret the results to provide furtherresults. The further processing step may analyse the results to providea DNA profile for the sample. The further processing step may provide anindication of a match between the sample and a database record of asample. The further processing step may be provided at a location remotefrom the instrument. The further processing step may be provided at alocation connected to the instrument, at least part of the time, by atelecommunications network. The further processing step may return tothe instrument and/or a computer, preferably within 200 m of the site ofthe instrument, the further processed results.

The results may be processed on the instrument to give processedresults. The processed results may extract from the results the signals,sections of signals or positions attributable to a characteristic beinganalysed for, such as an allele. The results and/or processed resultsmay be provided to the results output step.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present invention will now be described, byway of example only, and with reference to the accompanying drawings inwhich:

FIG. 1 is a schematic illustration of the stages involved in theconsideration of a sample from collection to results and illustrates thepositioning of the embodiments of the present invention in that context;

FIG. 2 is a schematic illustration of the key steps provided on or by aninstrument embodying the present invention;

FIG. 3 a is a front face view of part of a cartridge embodying thepresent invention;

FIG. 3 b is a table of dimensions and volumes for a cartridge accordingto the present invention, and components thereof;

FIG. 4 is a front face view of a further part of the cartridge of FIG. 3a and embodying further features of the present invention;

FIG. 5 a is a side view of the section of the cartridge of FIGS. 3 a and4 where in joins the electrophoresis cartridge section;

FIG. 5 b is a front view of the electrophoresis cartridge section shownin FIG. 5 a, with the section of the cartridge omitted;

FIGS. 6 a to 6 e are schematic illustrations of alternative arrangementsfor contacting the fluid and beads;

FIG. 7 is an illustration of an alternative structure for providingsample to the PCR chamber;

FIG. 8 is a front view of the electrophoresis cartridge section showingan alternative form of injector;

FIG. 9 is a schematic illustration of the parallel PCR chamberarrangement used in providing real time PCR and feedback of the results;

FIG. 10 a is an illustration of a closing valve used in the presentinvention;

FIG. 10 b is an illustration of an opening valve used in the presentinvention;

FIG. 11 shows an option for the archiving of a part of the samplehandled;

FIG. 12 is a schematic front view of one embodiment of the instrument;

FIG. 13 is a side view showing the insertion of the cartridge into theinstrument;

FIG. 14 is a schematic of the light source, optics and detector setupfor the electrophoresis section of the instrument;

FIG. 15 is an electropherogram showing the variation in signal from thedetector setup with time;

FIG. 16 is a schematic of an example of a system for detectingfluorescence;

FIG. 17 is a plot of LED spectrum, light reflected, and residual LEDlight over a range of wavelengths;

FIG. 18 is a plot of power of the LED-module over time;

FIG. 19 is an illustration showing beam shape and size as measured bythe laser camera;

FIGS. 20 a and 20 b are plots of CCD signal v/s wavelengths for staticfluorescence measurements; and

FIG. 21 is a plot of CCD signal v/s time for dynamic fluorescencemeasurements;

FIG. 22 is an illustration of a PCR chamber according to a furtherembodiment;

FIG. 23 is an illustration of the position of stacked Peltier effectdevices;

FIG. 24 is an illustration of an embodiment for loading a CE channel

FIG. 25 is an illustration of a further embodiment for loading a CEchannel;

FIG. 26 is an illustration of a further embodiment of a PCR chamber;

FIG. 27 is a front face view of a cartridge according to an embodiment;

FIG. 28 a is a front face view of a cartridge according to a differentembodiment;

FIG. 28 b is a table of dimensions and volumes for the FIG. 28 acartridge;

FIG. 29 a is a perspective view of an embodiment of the instrument;

FIG. 29 b is a front view of the instrument of FIG. 29 a;

FIG. 29 c is a side view of the instrument of FIG. 29 a;

FIG. 30 is a perspective view of another instrument embodiment;

FIG. 31 a is an illustration of a carrier, cartridge and CE chipembodiment;

FIG. 31 b is an illustration of a detail of the carrier to cartridgeengagement;

FIG. 32 a is an illustration of a carrier to CE chip engagement;

FIG. 32 b is a cut away illustration of a part of the FIG. 32 aengagement;

FIG. 33 a is an illustration of the tube and cartridge connection;

FIG. 33 b is an illustration of the tube to CE chip connection;

FIG. 34 a is an illustration of the carrier being inserted into theinstrument;

FIG. 34 b is an illustration of the inserted carrier;

FIG. 35 a is an illustration of the cartridge and carrier in theinsertion form;

FIG. 35 b is an illustration of the cartridge and carrier in the useform;

FIG. 35 c is an illustration of the cartridge returned to the carrier;

FIG. 36 a is a perspective view of the position of the pair of calipers;

FIG. 36 b is a perspective view of the back of the pair of calipers;

FIG. 36 c is a plan view of the caliper structure in the open form;

FIG. 36 d is a plan view of the caliper structure in the closed form;

FIG. 37 a is a perspective view of the second support of the carrier andCE chip;

FIG. 37 b is a partial cut away illustration of the second support andCE chip;

FIG. 38 is a perspective view of the CE chip heater board;

FIG. 39 is a perspective view of an embodiment of the optics;

FIG. 40 a is a perspective view of the alignment structure;

FIG. 40 b shows the alignment structure of FIG. 40 a in the stowedposition;

FIG. 40 c shows the alignment structure of FIG. 40 a in the useposition;

FIG. 41 a shows three positions for an alternative PCR chamberembodiment;

FIG. 41 b shows two positions for a further PCR chamber embodiment;

FIG. 41 c shows three positions for a still further PCR chamberembodiment;

FIG. 42 a shows a CE chip embodiment;

FIG. 42 b shows a detail of the CE chip of FIG. 42 a;

FIG. 43 shows an approach to loading sample to the CE step;

FIG. 44 shows an alternate approach to loading sample to the CE step;

FIG. 45 shows a further alternative for loading sample to the CE step

FIG. 46 shows a further embodiment of a PCR chamber.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a variety of cases it is desirable to be able to analyse a biologicalsample to obtain information on the sample and/or one or more componentsof the sample. Such cases include medical diagnostics, for instance tolook for disease markers, and forensic science, for instance toestablish a DNA profile.

At present, such analyses are conducted by highly trained scientists ina laboratory environment. This means that a significant amount of effortand experience goes into the handling of the samples, the use of theanalysis equipment and the formulation of the conclusions reached.However, the need to convey the sample to a laboratory environment andthen receive the results back from the laboratory environment introducesa potential time delay between obtaining the sample and obtaining theresults thereon. The need to use a laboratory environment and highlytrained scientists potentially adds to the time required, as the supplyof such people and resources is limited. The need to use a laboratoryenvironment and highly trained scientists potentially adds to the costas there are capital and running costs associated with such facilitiesand the scientists.

If fewer laboratory style environments are to be used for the analysisor the staff used are less specialised, then there is the potential forproblems with the analysis, unless a proper and reliable system isprovided.

The present invention has amongst its potential aims to enable analysisof samples at a greater variety of locations and/or non-laboratory typelocations. The present invention has amongst its potential aims toenable analysis by personnel having a lower level of training and/orexperience. The present invention has amongst its potential aims toenable lower cost and/or faster analysis of samples. The presentinvention has amongst its potential aims to enable greater use and/ormore successful use of analysis by law enforcement authorities.

Many of the concepts and issues to be addressed by the invention arebest understood by way of the following examples. It should be noted,however, that these examples are by their very nature detailed andexhaustive, and that benefits from the present invention arise even whenonly small sections of the examples are implemented in other embodimentsof the present invention.

The various embodiments and examples explain the invention initially inthe context of a reference sample; that is a sample collected from aknown individual under controlled conditions. An example of a referencesample would be a sample collected by a swab from the buccal cavity of aperson who has been arrested, the sample being collected at a policestation. The invention is also suited to casework samples; that is asample collected from a location from an unknown individual undernon-controlled conditions. An example would be a spot of blood collectedby a swab from a crime scene, with the source of the blood unknown.Where the differences between reference samples and casework sampleshave an impact on the preferred forms of the instrument, cartridge andmethods, the casework sample embodiments are separately described.

The substitution of one or more components by one or more differentcomponents or different arrangements of components is also envisagedwhere particular conditions or issues arise. Again, after the discussionof the reference sample and casework sample contexts for the instrument,these alternatives are described.

As a starting point, it is useful to establish the context of theinstrument, cartridge and methods of use in the overall context in whichthey may be used, by way of example. Thus in FIG. 1 there is a schematicof the overall process into which the present invention fits. Thisoverall process includes a sample 1 which is gathered in a samplecollection stage 3. This is followed by a sample preparation stage 5. Inthe subsequent sample loading stage 7, a prepared cartridge 9 is loadedwith the collected and prepared sample 1. The next stage is thecartridge installation stage 15 in which the cartridge 9 is introducedto the instrument 11. The instrument 11 also receives various inputs 13at the sample loading stage 7 and/or at the cartridge installation stage15 and/or subsequently.

The structure and processes performed within the instrument 11 andcartridge 9 are described further below in the context of FIG. 2.

Once the instrument 11 has completed these stages and achieved theanalysis, the next stage is the results stage 17. This is followed byone or more output stages 19, and potential further stages 21 whichintegrate the analysis into the criminal justice system of thatjurisdiction. A wide range of possible links between the various outputstages 19 and further stages 21 may be possible, with some being linkedto just one stage and others be the result of multiple such stagesand/or combinations thereof.

An output stage 19 may include the transmission of the results from theinstrument to a remote location for processing. The processing may beperformed using complex software and/or hardware tools, before the finalresults are returned to the instrument 11 or to another computer.Processing the results at a remote location may be preferably in termsof the size, cost or complexity of the software/hardware needed toperform the processing thus only being provided at a limited number oflocations, rather than a part of each instrument.

The following description of the operation of the instrument 11, in agenerally sequential manner, provides full details of the key instrumentstages and their interrelationship.

Referring to FIG. 2, the instrument has a sample receiving step 200,sample preparation step 202, sample amplification step 204,electrophoresis step 206 and analysis step 208 and data communicationstep 210.

In the sample receiving step 200, the sample 1 is transferred from asample storage and/or processing stage 5, which is outside of thecartridge 9 and instrument 11, to a location on the cartridge 9.

The initial collection device is frequently a swab. The swab is used topick up the sample 1 from an article or substrate.

In the sample preparation step 202, the key components within the sampleare contacted with the reagents and/or components intended to preparethe sample for the subsequent steps. In this embodiment, the samplepreparation step 202 contacts the sample with beads to retain the DNAand recover it, whilst the other components which are not to berecovered flow through and away. The sample preparation step 202 alsoincludes contact with a wash agent to improve the separation of the DNAfrom the other components. The wash agent flows through the chamberholding the beads and retained DNA and flows to a further chamber, awaste chamber. The wash agent is followed by an elution agent to releasethe DNA from the beads for the subsequent steps.

In the sample amplification step 204, the DNA is contacted withamplification reagents and provided with the conditions necessary toachieve amplification through PCR.

In the electrophoresis step 206, the amplified DNA is conveyed to astart point for a mobility based separation within a capillary. Anelectric field is then used to separate the complex DNA amplicons intodifferent size clusters.

In the analysis step 208, the channel is inspected to establish therelative position and hence size of elements detected in the capillary.This is achieved by an excitation light source, fluorescent markersassociated with the elements to be detected and suitable optics todetect the fluorescent light resulting.

In the data communication step 210, the instrument compiles thenecessary data packet for transmission and transmits it to a remotelocation for consideration. The data packet includes information on theelectrophoresis results, sample identity and other information. Theanalysed results may be received by the instrument as part of the datacommunication step 210.

Some data processing may be performed on the instrument itself, forinstance to deconvolute the analysis results to indicate the peaksindicative of alleles present.

The instrument can be provided in a format which considers a singlesample at a time, or can be provided in a format which considersmultiple samples at a time. The multiple samples may each be run onseparate cartridges, but modified cartridges which handle multiplesamples are possible. The handling of multiple cartridges is beneficialin allowing a single set of controllers, power supplies, optics and thelike to consider multiple samples, with reduced capital costs.

Cartridge

Key to the operation of the instrument is a disposable, single usecartridge 9. This cartridge 9 is intended to only process and providethe results for analysis on a single occasion. The disposable nature ofthe cartridge 9 places a number of constraints on the cartridge 9 interms of the materials which can be used, because of the need to keepmanufacturing, assembly or purchase costs low.

The detailed layout of the cartridge 9 is now described. Later, adescription of the sequence of operation of the elements which make upthe cartridge is provided.

FIG. 3 a is an illustration of that part of the sample receiving step200 provided on the cartridge 9, the whole sample preparation step 202and the whole sample amplification step 204. The subsequent steps andtheir respective pasts of the cartridge 9 are illustrated separately.

FIG. 3 b provides details of the volumes of the various chambers used,the depths (into the page in effect) for the various components and theoverall dimensions of this part of the cartridge 9.

The cartridge 9 is provided with a sample introduction chamber 302connected to a channel 304 leading to the outside of the cartridge 300.This forms those parts of the sample receiving step 200 provided on thecartridge 9.

The sample preparation step 204 follows. To provide this, the sampleintroduction chamber 302 is connected to a pumping fluid channel 306 andhence to a first electrochemical pump 308. The sample introductionchamber 302 has an outlet channel 310 which passes valve 312 andprovides an inlet to purification buffer chamber 314. Valve 312 isinitially open.

Purification buffer chamber 314 is connected via channel 316 to beadstorage chamber 318. The bead storage chamber 318 is connected viachannel 320 to initial mixing chamber 322. The outlet channel 324 frominitial mixing chamber 322 is blocked by closed valve 326, but a ventchannel 328 is open because valve 330 is open initially.

The outlet channel 324 leads past valve 326 to a first further mixingchamber 332 and then through channel 334 to second further mixingchamber 336. The outlet 338 from the second further mixing chamber 336leads past valve 340 to incubation chamber 342, where bubble mixingassists the DNA to bead binding process.

The incubation chamber 342 has a vent channel 344 provided with valve346 and an outlet channel 348 which is initially closed by valve 350.The incubation chamber 342 is also provided with a pumping fluid inletchannel 352 which passes valve 354 and is connected to secondelectrochemical pump 356.

The outlet channel 348 from the incubation chamber 342 leads to capturechamber 358 where the beads and hence bound DNA are collected. Thecapture chamber 358 is provided with a first vent channel 360 whichpasses first valve 362 and second valve 364. The capture chamber 358 isalso provided with a second vent channel 366 which passes first valve368 and second valve 370.

Also connected to capture chamber 358 is wash buffer channel 372. Thewash buffer channel is connected to first valve 374 and second valve 376and leads from second electrochemical pump 356 through wash bufferchamber 378 to the capture chamber 358.

Also connected to capture chamber 358 is an elution liquid channel 380.The elution liquid channel 380 is connected to first valve 382, elutionliquid storage chamber 384, second valve 386 and back to thirdelectrochemical pump 388.

The capture chamber 358 has a wash outlet channel 390 which splits intoa first wash outlet channel section 392 which passes valve 394, and intoa second wash outlet channel section 396 which passes valve 398. Afterpassing their respective valves 394, 398, the first wash outlet channelsection 392 and second wash outlet channel section 396 rejoin oneanother to form further wash channel 400. The further wash channel 400leads past valve 402 into waste chamber 404. The waste chamber 404 isvented along vent channel 406 past valve 408. These elements provide thesample preparation step 202.

To provide the sample amplification step 204, capture chamber 358 isalso provided with elution outlet channel 410 which leads past valve 412and past valve 414 and into PCR chamber 416. The outlet channel 418 fromthe PCR chamber 416 leads past valve 420 into archive chamber 422. Thearchive chamber 422 is vented through vent channel 424. The role of thearchive chamber 422 is described further below.

Provided within the PCR chamber 416 is a bead loaded with the reagents,a multimix, needed for the PCR process. The reagents/multimix includeprimers dNTPs and PCR reaction mix, including Tris buffer, MgCl₂, NaCland BSA. These reagents are released into the sample once it contactsthe bead in the PCR chamber 416 and the temperature is raised aboveambient temperature.

The above circuit overall, is sufficient to receive, retain, wash, eluteand perform PCR on the sample, as well as storing the waste from theprocess and an archive of the PCR product.

Subsequently, the arrangement shown in FIG. 4 can be used to transferthe now amplified DNA from the PCR chamber 416 into the electrophoresisstep 206.

In FIG. 4, the PCR chamber 416 is the same PCR chamber 416 which wasillustrated in FIG. 3 and described above. Other features were omittedfrom FIG. 3 to improve the clarity of that Figure.

Leading from the PCR chamber 416 is a denaturing feed channel 500 whichis connected to an amplified material mixing chamber 502. The amplifiedmaterial is pumped from PCR chamber 416 by the action of fourthelectrochemical pump 504 which is connected to channel 506, hence todenaturing reagent storage chamber 508 and through channel 510 to thePCR chamber 416. Formamide is provided in the denaturing reagent storagechamber in the preferred form.

These components are isolated from the PCR chamber 416 during the sampleamplification step 204 by closed valve 512 and closed valve 514. Bothvalve 512 and 514 are opened and valves 516 and 518 are closed to conveythe amplified material away from the PCR chamber 416.

From the denaturing feed channel 500, the amplified material anddenaturing reagents enter the first amplified material mixing chamber502, pass through channel 520, into second amplified material mixingchamber 522, through channel 524 and into third amplified materialmixing chamber 526. Whilst the third amplified material mixing chamber526 fills, valve 528 is shut and vent 530 is open. An overall volume of45:1 is provided, 5:1 from the PCR chamber and 40:1 from the denaturingreagent storage chamber 508.

The amplified material is held in the third mixing chamber 526 for thenecessary time and at the necessary temperature to complete thedenaturing process. Once this has been achieved, the valve 528 is openedand further pumping by the fourth electrochemical pump 504 pumps thedenatured material to the electrophoresis step inlet 532. At the inlet532, the denatured material passes out of the plane of the cartridge 9and to the electrophoresis cartridge section behind. Once past throughthe inlet 532, valve 534 is shut to isolate the cartridge 9 from theelectrophoresis cartridge section 600.

The overall result of this structure is the pumping of the amplified DNAto a start point for the electrophoresis step 206.

The transfer from PCR to CE steps is provided in a way which allows easyintegration of the steps, does not impact upon the temperature andpressure stability required in PCR and achieves minimal sample lossduring transfer. Automated mixing of the sample and size standardsduring transfer and possibilities for pre-concentrating the samplebefore CE are also rendered possible.

The overall configuration of the electrophoresis step 206 can be seen inthe side view of FIG. 5 a and front view of FIG. 5 b.

The inlet 532 leads from the plane of the cartridge 9, through into theplane of the electrophoresis cartridge section 600. Here, the inlet 532leads into the top section 602 of an electrophoresis feed reservoir 604.The top section 602 is empty, but the lower section 606 is provided withthe gel 608 which also fills the capillary 610. The sample is pumpedinto the electrophoresis feed reservoir 604 by a fourth electrochemicalpump, not shown.

Sample flow from the reservoir 604 into the correct position within thecapillary 610 is achieved using electrophoresis as the transportmechanism.

In this embodiment, the injector structure provided within the capillarycartridge section 600 is a double T injector. This includes a firstelectrode location 612, second electrode location 614 provided at theother end of the long capillary 616 in which the size based separationis achieved. A third electrode location 618 and fourth electrodelocation 620 are provided in side arms 622 and 624 respectively. Theside arms are offset relative to one another, with side arm 624 furthertowards the second electrode location 614, than the side arm 622.

Initially, sample is drawn from the liquid phase in the reservoir 604through the interface with the gel provided in the reservoir 604 andhence into the gel by a voltage applied to the electrode present at thethird electrode location 618. Once the sample has been drawn past thefourth electrode location 620, a voltage is also applied to theelectrode at the fourth electrode location. Generally, the electrode atthe third electrode location may be at a voltage of 600V and theelectrode at the fourth electrode location may be at a voltage of 200V.The voltage may be floating for the electrodes at the first 612 andsecond 614 electrode locations.

This situation results in sample being drawn along side arm 624, alongthe section 626 and into side arm 622, such that sample is present inthe two side arms 622 and 624 and the section 626 of the capillary 616.

This gives the plug of sample upon which the electrophoresis's to act inthe section 626.

To reduce the cost of the electrodes used, consistent with the cartridgebeing single use, platinum coated, gold coated, carbon, nickel and otherlower cost electrodes may be used.

Once positioned, the separation voltages are applied: 1500V at theelectrode at the second electrode location 614; 0V at the electrode atthe first electrode location 612; and 200V at the electrodes present atthe third electrode position 618 and fourth electrode positions 620.

The capillary 616 is filled with a gel matrix which preferentiallyretards the speed of progress of elements within the DNA as their sizeincreases. The result is a size based separation of the elements, withthe faster elements reaching the detection location 626 first and theslowest reaching the detection location 628 last. The different times atwhich the signals are generated and form the electropherogram indicatethe size of the element behind that signal.

It is possible to assist in the interpretation of the unknown elementsizes by using a size standard within the capillary. This is providedwith a different dye colour or otherwise rendered distinct. The methodset out in U.S. patent application No. 61/096,424, the contents of whichare hereby incorporated by reference, offers approaches for determiningthe sizes of the unknowns from the size standard.

The setup and operation of the light source, optics and detector isdescribed in detail below.

Other embodiments of the cartridge have also been developed.

As shown in FIG. 27, the cartridge 27-01 has been modified by providingthe electrochemical pumps 27-03, 27-05, 27-07, 27-09 with connectionsbetween the wires leading to the electrodes in the pumps and the powersource not shown of the Pogo™ pin type. The pins 27-11 are spring loadedin the recesses of the cartridge 27-01 and in use contact similar springloaded pins (not shown) on the other side of the cartridge to instrumentinterface. A reliable electrical contact is thus provided and thecartridge is more robust against damage during storage, installation anduse than designs in which the wires for the electrochemical pumpsprotruded from the side of the cartridge.

The form shown in FIG. 27 also features guide holes 27-13 which are usedin the alignment of the cartridge and instrument, as described in moredetail below.

A preferred embodiment of the cartridge is shown in FIG. 28 a. This isan illustration of that part of the sample receiving step 200 providedon the cartridge 28-09, the whole sample preparation step 202, the wholesample amplification step 204, the whole sample denaturation step andthe feed to the capillary electrophoresis step 206.

FIG. 28 b provides details of the volumes of the various chambers used,the depths (into the page in effect) for the various components and theoverall dimensions of this part of the cartridge 28-09.

The cartridge 28-09 is provided with a sample introduction chamber28-302 connected to a channel 28-304 leading to the outside of thecartridge 28-09. This forms those parts of the sample receiving step 200provided on the cartridge 28-09.

The sample preparation step 204 follows. To provide this, the sampleintroduction chamber 28-302 is connected to a pumping fluid channel28-306 and hence to a first electrochemical pump 28-308. The sampleintroduction chamber 28-302 has an outlet channel 28-310 which passesvalve 28-312 and provides an inlet to bead storage chamber 28-318. Valve28-312 is initially open.

The bead storage chamber 28-318 has an outlet channel 28-316 leading tobinding buffer storage chamber 28-314. This sequence of chambers isreversed compared with the FIG. 3 a embodiment. The binding bufferstorage chamber 28-314 has an outlet channel 28-320 which leads tomixing/purification chamber 28-322.

Mixing/purification chamber 28-322 is connected via channel 28-324through valve 28-326 and via channel 28-500 to first further mixingchamber 28-332. The outlet channel 28-324 from mixing/purificationchamber 28-322 is blocked by closed valve 28-326, but a vent channel28-328 is open because valve 28-330 is open initially.

The outlet channel 28-324 leads past valve 28-326 to a first furthermixing chamber 28-332 and then through channel 28-334 to second furthermixing chamber 28-336. The outlet 28-338 from the second further mixingchamber 28-336 leads past valve 28-340 to incubation chamber 28-342,where bubble mixing assists the DNA to bead binding process. Theincubation chamber 28-342 may be actively heated or may simply providethe necessary dwell time and/or other binding conditions needed.

The incubation chamber 28-342 has a vent channel 28-344 provided withvalve 28-346 and an outlet channel 28-348 which is initially closed byvalve 28-350. The incubation chamber 28-342 is also provided with apumping fluid inlet channel 28-352 which passes valve 28-354 and isconnected to second electrochemical pump 28-356.

The outlet channel 28-348 from the incubation chamber 28-342 leads tocapture chamber 28-358 where the beads and hence bound DNA arecollected. The capture chamber 28-358 is provided with a first ventchannel 28-360 which passes first valve 28-362 and second valve 28-364.The capture chamber 28-358 is also provided with a second vent channel28-366 which passes first valve 28-368 and second valve 28-370.

Also connected to capture chamber 28-358 is wash buffer channel 28-372.The wash buffer channel is connected to first valve 28-374 and secondvalve 28-376 and leads from second electrochemical pump 28-356 throughwash buffer chamber 28-378 to the capture chamber 28-358.

Also connected to capture chamber 28-358 is an elution liquid channel28-380. The elution liquid channel 28-380 is connected to first valve28-382, elution liquid storage chamber 28-384, second valve 28-386 andback to third electrochemical pump 28-388.

The capture chamber 28-358 has a wash outlet channel 28-390 which splitsinto a first wash outlet channel section 28-392 which passes valve28-394, and into a second wash outlet channel section 28-396 whichpasses valve 28-398. After passing their respective valves 28-394,28-398, the first wash outlet channel section 28-392 and second washoutlet channel section 28-396 rejoin one another to form further washchannel 28-400. The further wash channel 28-400 leads past valve 28-402into waste chamber 28-404. The waste chamber 28-404 is vented along ventchannel 28-406 past valve 28-408. These elements provide the samplepreparation step 202.

To provide the sample amplification step 204, capture chamber 28-358 isalso provided with elution outlet channel 28-410 which leads past valve28-412 and past valve 28-414 and past valve 28-502 and into PCR chamber28-416. The outlet channel 28-418 from the PCR chamber 28-416 leads pastvalve 28-420 and past valve 28-504 and past valve 28-506 into archivechamber 28-422. The archive chamber 28-422 is vented through ventchannel 28-424. The role of the archive chamber 28-422 is as describedfurther above.

Provided within the PCR chamber 28-416 is a bead loaded with thereagents, a multimix, needed for the PCR process. The reagents/multimixinclude primers dNTPs and PCR reaction mix, including Tris buffer,MgCl₂, NaCl and BSA. These reagents are released into the sample once itcontacts the bead in the PCR chamber 28-416 and the temperature israised above ambient temperature.

The above circuit overall, is sufficient to receive, retain, wash, eluteand perform PCR on the sample, as well as storing the waste from theprocess and an archive of the PCR product.

The PCR part of the circuit has been moved to the upper section of thecartridge compared with the previous embodiments so as to present itphysically closer to the CE chip.

Subsequently, the further arrangement shown in FIG. 28 a can be used toprepare, denaturation step, and transfer the now amplified DNA from thePCR chamber 28-416 into the electrophoresis step 206.

Leading from the PCR chamber 28-416 is outlet channel 28-418. Thissplits after valves 28-420 and 28-504 into a denaturing feed channel28-550 and the channel leading to the archive chamber 28-422. Thedenaturing feed channel 28-550 is connected to a denaturation chamber28-552. The amplified material is pumped from PCR chamber 28-416 by theaction of fourth electrochemical pump 28-554 which is connected tochannel 28-556, hence to denaturing reagent storage chamber 28-558 andthrough valve 28-560 and channel 28-562 to the PCR chamber 28-416.Formamide is provided in the denaturing reagent storage chamber 28-558in combination with the size standards to be used in the capillaryelectrophoresis step.

These components are isolated from the PCR chamber 28-416 during thesample amplification step 204 by closed valve 28-502 and closed valve28-420. Both valve 28-502 and 28-420 are opened and valves 28-414 and28-506 are closed to convey the amplified material away from the PCRchamber 28-416 to the denaturation chamber 28-552. This is ventedthrough valve 28-564, with exit channel 28-566 closed by valve 28-568.

The amplified material is held in the denaturation chamber 28-552 forthe necessary time and at the necessary temperature to complete thedenaturing process. Once this has been achieved, the valve 28-568 isopened and further pumping by the fourth electrochemical pump 28-554pumps the denatured material to the electrophoresis step inlet 28-570.

At the inlet 28-570, the denatured material passes out of the plane ofthe cartridge 9 and through a tube to the electrophoresis cartridgesection behind. The overall result of this structure is the pumping ofthe amplified DNA to a start point for the electrophoresis step 206.

Details of the connection of the inlet 28-570 to the CE chip areprovided below.

Throughout the operations described above and in the sections thatfollow, various checks are made on operating conditions, componentperformance and successful operation so as to ensure the processing iscorrectly provided from start to finish. Errors or problems areindicated to the operator.

Cartridge Sequence of Operation

The sequence of operation, purely by way of example, applied to thecartridge shown in and described in relation to FIGS. 3 a and b is asfollows, with sample timings also given.

Time since Purpose and start (sec) Change notes 0.0 Incubation chamber358 - adjust temperature to 25° C. 0.9 Valve 312 - opening valve - heaton 31.5 First electrochemical pump 308 - on 73.3 Valve 330 - closingvalve - heat off 121.1 Valve 312 - opening valve - heat off 138.7 Firstelectrochemical pump 308 - off 187.8 Valve 326 - opening valve - heat on212.3 Valve 312 - opening valve - heat on 233.9 Valve 330 - closingvalve - heat off 236.0 First electrochemical pump 308 - on 324.3 Valve312 - opening valve - heat off 368.6 Valve 326 - opening valve - heatoff 370.4 Valve 346 - closing valve - heat on 401.0 Firstelectrochemical pump 308 - off 461.4 Valve 346 - closing valve - heatoff 653.4 Valve 350 - opening valve - heat on 655.1 Magnet - fieldapplied to chamber 656.4 Valve 326 - opening valve - heat on 684.5 Firstelectrochemical pump 308 - on 783.4 Valve 326 - opening valve - heat off804.1 Valve 394 - closing valve - heat on 815.4 Valve 340 - closingvalve - heat on 829.6 Valve 350 - opening valve - heat off 840.8Magnet - field removed from chamber 867.5 First electrochemical pump308 - off 894.2 Valve 394 - closing valve - heat off 944.5 Valve 368 -opening valve - heat on 975.5 Valve 340 - closing valve - heat off 977.2Second electrochemical pump 356 - on 1025.8 Valve 354 - closing valve -heat on 1036.2 Valve 368 - opening valve - heat off 1050.8 Secondelectrochemical pump 356 - off 1079.7 Valve 324 - opening valve - heaton 1080.6 Valve 368 - opening valve - heat on 1116.3 Valve 354 - closingvalve - heat off 1118.0 Second electrochemical pump 356 - on 1181.3Valve 370 - closing valve - heat on 1196.4 Valve 368 - opening valve -heat off 1228.3 Valve 324 - opening valve - heat off 1233.9 Secondelectrochemical pump 356 - off 1244.2 Valve 398 - opening valve - heaton 1249.4 Valve 324 - opening valve - heat on 1271.8 Valve 370 - closingvalve - heat off 1273.1 Magnet - field applied to chamber 1284.7 Secondelectrochemical pump 356 - on 1328.6 Valve 324 - opening valve - heatoff 1333.8 Valve 402 - closing valve - heat on 1334.7 Valve 408 -closing valve - heat on 1379.9 Valve 398 - opening valve - heat off1383.8 Magnet - field removed from chamber 1393.9 Second electrochemicalpump 356 - off 1419.5 Valve 362 - opening valve - heat on 1435.4 Valve402 - closing valve - heat off 1465.1 Valve 408 - closing valve - heatoff 1466.0 Second electrochemical pump 356 - on 1474.6 Valve 374 -closing valve - heat on 1493.6 Valve 362 - opening valve - heat off1501.8 Valve 382 - opening valve - heat on 1504.8 Valve 362 - openingvalve - heat on 1508.7 Second electrochemical pump 356 - off 1531.9Third electrochemical pump 388 - on 1578.8 Incubation chamber 358 -adjust temperature to 60° C. 1585.0 Valve 374 - closing valve - heat off1586.6 Valve 362 - opening valve - heat off 1588.5 Valve 364 - closingvalve - heat on 1633.3 Valve 382 - opening valve - heat off 1640.4 Thirdelectrochemical pump 388 - off 1679.0 Valve 364 - closing valve - heatoff 1881.0 Valve 412 - opening valve - heat on 1882.9 Valve 382 -opening valve - heat on 1906.2 Magnet - field applied to chamber 1914.9Third electrochemical pump 388 - on 1952.3 Incubation chamber 358 -adjust t to 25° C. 2010.0 Third electrochemical pump 388 - off Magnet -field removed from chamber Valve 382 - opening valve - heat off Valve412 - opening valve - heat off 2017.3 Valve 420 - closing valve - heaton Isolate PCR Valve 414 - closing valve - heat on chamber 2173.3 Valve420 - closing valve - heat off Valve 414 - closing valve - heat off2185.0 Incubation chamber temperature control - off

Cartridge Alternatives

There are a variety of alternatives for the various components withinthe cartridge and/or their operation. Some of these are now described,by way of example only.

1) Bead Handling

As described above, the cartridge makes use of a bead storage chamber318 from which the beads are washed in operation. This washing actionprovides contact between the sample, reagents and the beads. Mixingresults in the beads taking up the DNA in the sample and retaining it.Subsequent retention of the beads allows the DNA to be separated fromthe rest of the sample and allows washing stages to improve further thisseparation.

It is important to ensure that the beads are displaced from theirstorage location, such that the beads are available, in contact with therelevant liquids, to perform their task. Modifications to the manner inwhich the beads are stored and/or dispensed can assist in this. Thebeads may be stored away from the cartridge. They may be introduced tothe cartridge to prepare it for use.

Firstly, it is possible to provide a dispersant together with the beadsso as to keep them dispersed and hence more easily collected and carriedby the fluid flow. This can help prevent blockages and/or agglomerationsof beads. Different dispersants and/or variations in the amount providedcan be used to tailor this.

Secondly, it is possible to provide the beads in a series of beadstorage chambers, rather than in a single chamber. FIG. 6 a illustratesone such arrangement, where the beads are split into three groups, eachin its own chamber 700. In this way, the contact between the fluid andthe beads is staggered and a compacted mass of beads is avoided on thelead edge of the fluid. A variation on this is provided in FIG. 6 b,where a first bead storage chamber 700 a is separated from the secondbead storage chamber 700 b by a mixing chamber 702.

Thirdly, the contact can be provided with a thin chamber 704 whereby thetransition of the fluid from the thing channel 706 into the chambercauses non-laminar flow and hence improved mixing. The provision of thebeads spread along the length of the chamber 704 also means that they donot contact the fluid all at the same time.

Fourthly, the flow direction and/or chamber design can be modified toencourage displacement of the beads from their storage position into amixed form with the fluid. Thus in the FIG. 6 d form, the fluid entersthe chamber 700 in one bottom corner 708 and displaces, arrows, thebeads resting in that part. A swirling flow within the chamber 700 givesmixing, before the fluid and bead mixture exits the chamber 700 throughthe other bottom corner 710.

Fifthly, the beads can be stored in a side arm 712 or other form ofpassage. As the flow of fluid passes through thin chamber 714 and pastthe junction 716 with the arm 712, a force is applied behind the mass ofstored beads in the side arm 712. This forces the mass of stored beadstowards and into the junction 716 where they gradually contact and areswept away by the fluid flow. Gradual dispersal of the beads into thefluid is provided. The motive force behind the beads can be provided bya similar structure to that used to move material in the context of theclosing valves described herein.

2) PCR Chamber Filling

In the above system, the amount of the processed sample which is madeavailable to the PCR stage is controlled by the relative height of theoutlet from the PCR chamber to the archive chamber leading to overflowof excess sample into the archive chamber. This results in a PCR chamberwhich is not completely full of sample during PCR. As PCR involvesheating of the sample, evaporation and/or condensation of part of thesample may occur at a location outside of the PCR chamber. This canreduce the reagents present in the PCR chamber and hence reduce theefficiency of the PCR stage.

In an alternative form, the PCR chamber is entirely filled with thesample before PCR is started. This is achieved using the arrangement ofFIG. 7 where the majority of the components have the same structure andfunction as shown in the FIG. 3 and FIG. 4 description. The differencesare in the section around the PCR chamber 416.

In this alternate form, the PCR chamber 1416 is fed material alongchannel 1413. Initially, the path of least resistance to this fluid flowis through the PCR chamber 1416, along channel 1500, past opened valve1502 and onto vent 1504. The vent 1504 is hydrophobic and so allows thepassage of the air displaced from the PCR chamber 1416 and channel 1500by the material's advance. Once the fluid reaches the vent 1504,however, the path of least resistance changes and further flow occursalong channel 1418 past valve 1428 and into archive chamber 1422, whichis provided with vent 1424. By this time, the PCR chamber 1416 iscompletely full of liquid and hence the volume of the liquid subjectedto PCR is guaranteed.

As before, the valves around the PCR chamber 1416 are closed during theamplification itself, so as to isolate the PCR chamber 1416.

In a third alternative, the configuration shown in FIG. 22, the PCRchamber 22-01 is along channel 22-03. Initially, the path of leastresistance to this fluid flow is through the inlet 22-05 to the PCRchamber 22-01. Once the PCR chamber 22-01 has filled, the liquidoverflows through exit 22-07 into channel 22-09 which is a continuationof channel 22-03. Further fluid flow simply by-passes the PCR chamber22-01 and flows through channel 22-03 and then channel 22-09. To controlthe flow correctly, the dimension A of the inlet 22-05 is greater thanthe dimension B of the outlet 22-07. The dimension is preferably greaterin terms of the cross-sectional area, perpendicular to the direction offlow. The complete filling of the PCR chamber 22-01 ensure the volume ofthe liquid subjected to PCR is guaranteed.

Various shapes are possible for the PCR chamber. FIG. 26 provides anexample in which the PCR chamber 26-01 is formed as smooth as possible.This assists with full fluid contact with the surfaces and hencecomplete and accurate filling of the PCR chamber 26-01. The sample flowsalong channel 26-03 and enters the PCR chamber 26-01 via inlet 26-05provided towards the bottom of the PCR chamber 26-01. The sample fillsthe PCR chamber 26-01 before overflowing through outlet 26-07 providedtowards the top of the PCR chamber 26-01 and into channel 26-09.

In the embodiment of FIG. 46, a variation on the above principle isprovided. The flow to the PCR chamber 46-100 passes along channel 46-102and past valve 46-104. The channel 46-102 turns as it approaches thechamber 46-100 and provides inlet channel 46-106. The natural flow isalong this route. As the flow progresses, the PCR chamber 46-100 fills,with the gas exiting through outlet channel 46-108. The outlet channel46-108 has a similar configuration to inlet channel 46-106, but thecross-sectional area of the outlet channel 46-108 is much smaller thanthat of the inlet channel 46-106. As a result, when the liquid reachesthe outlet channel 46-108, the flow resistance increases greatly andflow is redirected along the by-pass channel 46-110 in preference. Boththe outlet channel 46-108 and the by-pass channel 46-110 lead past valve46-112 to exit channel 46-114. The Peltier effect device heats the areawithin the dotted lines and so ensures that as much of the space betweenthe two valves, 46-104 and 46-112 is heated so as to minimise anycondensation within that space.

3) Sample Concentration Before Capillary Electrophoresis

In some instances, it may be helpful to increase the concentration ofthe sample prior to its use in the electrophoresis step and/or to reducethe size of the sample as it is injected.

Once suitable approach for doing so is set out in European patentpublication no 1514100, the contents of which are incorporated herein byreference. This technique uses careful balancing of the electrophoreticvelocity of the DNA and the opposing electroosmotic velocity toconcentrate the DNA at the liquid to gel interface. A change inconditions can then be used to drawn the concentrated DNA into theelectrophoresis step as a concentrated and small sample.

Another option is hydrodynamic stacking. This is based upon thevariation in the flow velocity between sample and the location fromwhich the size based separation starts, for instance through the use ofadjustments to conductivity, buffer components, pH and the like. Anexample of such an approach is field amplified sample stacking, FASS.This provides higher electric fields in the lower conductivity zonesthan in the higher conductivity zones. The sudden potential drop at theinterface between the two zones causes sample stacking there.

Mechanical pre-concentration is also a possibility. Packed beds,nanochannels, immobilised polymers and membranes all offer thepossibility of trapping and concentrating the sample. Electro-elution,where by the release of the sample is caused by the application of anelectric potential to a membrane, is one possibility.

A combined technique approach to pre-concentration may be particularlybeneficial. Such an approach is shown in FIG. 24, in the case of CEchannel being in the same plane as the rest of the cartridge, and FIG.25, in the case of the CE channel not being in the same plane as therest of the cartridge.

As illustrated, the combined flow 24-01, 25-01 of DNA containing sampleand formamide pass valve 24-03, 25-03 and then reach a junction 24-05,25-05. The Y-shaped junction brings the combined flow 24-01, 25-01 intoproximity with the running buffer flow 24-07, 25-07 in channel 24-08,25-08. These flows cross the CE channel 24-09, 25-09 and any excesspasses to chamber 24-11, 25-11. The left-hand detail shows theconstruction present at the intersection of the CE channel 24-09, 25-09and the channel 24-08, 25-08.

In the FIG. 24 form, the stacking interface 24-11 is provided betweenthe combined flow 24-01 and buffer flow 24-07. The electric potential isprovided by electrode 24-13. The second stacking function is provided bythe membrane 24-15 provided between the buffer flow 24-07 and the CEchannel 24-09.

In the FIG. 25 form, the stacking interface is similarly provided.

4) Alternative Electrophoresis Channel Configuration

In the embodiment described above, the injector is of the double T type.As an alternative, it is possible to use a cross-channel injector, asshown in FIG. 8.

In this case, the reservoir 604, channel 610 and other parts leading tothe fourth electrode location 620 are the same. The arm 624 providedwith the fourth electrode location 620 and the arm 622 provided with thethird electrode location 618 are aligned on a common axis and at 90° tothe main capillary 616.

The sample is drawn towards the electrode at the third electrodeposition 618 by the application of a voltage. To prevent dispersion ofthe sample into the main capillary, towards the first 612 and/or second614 electrode locations, a voltage is applied to the electrode at thefirst electrode location 612 and to the electrode at the secondelectrode location 614. This has the effect of pinching the part of thesample at the intersection of the main capillary 616 and the arms 622,624, and maintaining the minimal size of the plug which is then used inthe capillary electrophoresis.

A further electrophoresis channel configuration is shown in FIG. 43. Inthis case, the sample flows along channel 43-100 from inlet 43-102 tooutlet 43-104. A potential difference is applied between locations A andB. This draws the DNA in the sample towards the membrane 43-106. Themembrane is sized, 10-14 kDa cutoff, to retain the DNA. The separationmatrix is then flowed into the channel 43-100; UV activation may beprovided, as discussed elsewhere. The same buffers at location A, B andin the matrix are then provided for the electrophoretic separation to beprovided through the application of a potential difference between A andB.

The polarity may be provided in the reverse direction before the CE run,for instance to ensure the buffer extends from A to B. DNA is not lostas the flow will maintain it on the membrane 43-106.

Between loading to the membrane 43-106 and the CE separation, it ispossible to introduce a variety of reagents/buffers into locations Aand/or B and/or the channel 43-100 to assist in purifying the DNA and/orto optimise CE conditions, for instance through removal of excess saltsand/or unincorporated PCR primers. Both locations A and B have their owninlets and outlets for this purpose.

A still further configuration is shown in FIG. 44. In this case, againthe sample flows through channel 44-100 from inlet 44-102 to outlet44-104. A potential difference between A and B is used to attract andretain the DNA on a membrane 44-106. By swapping to an electrolyte flowthrough channel 44-100 and changing the potential difference it ispossible to load the DNA to the matrix in main channel 44-108. The CEcan then be performed.

Again one or more cleaning or condition controlling steps may beprovided before CE is conducted.

A yet further configuration is shown in FIG. 45. In this case, the arm45-100 leading the sample into the main channel 45-102 where CE isperformed extends downwards, at least partially aligned with gravity.The arm 45-104 leading away from the main channel 45-102 extends upward,at least partially aligned with gravity. In this way gravitation effectspromote retention within the main channel 45-102, rather thanencouraging flow away from it and into another arm.

5) Cartridge Variant for Real Time PCR Performance

In the cartridge 9 described above, the cartridge 9 is being used toconsider a reference sample. In this alternative embodiment, the changesto the cartridge 5009 beneficial to the consideration of a caseworksample are considered.

A major difference between a casework sample and a reference sample isthat whilst the amount of DNA recovered in a reference sample has adegree of consistency, and is of a high level, this is not the case fora casework sample. The manner in which the sample is left, the passageof time, the collection process and other factors can all result in theamount of DNA in a casework sample being unpredictable, and often lower,than desired.

To counteract this, the casework sample processing seeks to ensure thatthe amount of DNA arising from the amplification process is withincertain bounds.

To do this, the casework sample provides for parallel processing of thesample, particularly in terms of the sample amplification step 204.

The sample receiving step 200 and sample preparation step 202 arebasically the same as previously described. The difference comes in thesample amplification step 206.

The channel 5410 containing the eluted DNA from the beads held in theincubation chamber 5358 leads to a junction 5700 where the flow is splitinto two separate streams 5702, 5704.

The first stream 5702 passes into a PCR chamber 416 of the typepreviously described (and is not illustrated further). The subsequenthandling of this by the cartridge 9 is as described above, save for thepossible changes in the sample amplification conditions/durationdescribed shortly.

The second stream 5704 passes into a second separate PCR chamber 5706.This second PCR chamber 5706 contains a bead provided with a coatingcontaining the necessary regents for PCR and for a quantificationanalysis.

During processing, PCR is advanced in the PCR chamber 416 and in thesecond PCR chamber 5706, in parallel. After a given number of PCR cyclesfor the second PCR chamber 5706, the contents of the second PCR chamber5706 are considered to establish the quantity of DNA which has beengenerated by the PCR cycles up to that point. This can be equated to theamount of DNA present within the original sample and hence the amount ofDNA the PCR chamber 416 is working on. As a result of thequantification, the PCR conditions and/or cycle number for the PCRchamber 416 can be varied to optimise the quality of amplificationproduct.

Further details on the operation of such a system and the use of thisfeed back are to be found in 61/026,869, the contents of which areincorporated herein by reference, particularly as they relate to theparallel conduct of PCR and the use of the results from one PCR tocontrol and/or modify the conduct of the other PCR.

Suitable reagents include the Plexor HY kit available from Promega Inc,2800 Woods Hollow Road, Madson, Wis. 53711, USA and Quantifiler® Duo DNAquantification kit available from Applied Biosystems, Foster City,Calif., 944404, USA.

To establish the quantity of DNA present, it is necessary to interrogatethe sample using an excitation light source and then quantify the amountof light arising. To do this, light from a light source is conveyed tothe second PCR chamber 5706 and focussed thereon using a lens system.The excitation light interacts with the dye(s) associated with thesample. The fluorescent light generated is detected and is proportionalto the quantity of DNA present.

The light source used could be the same light source as is used for theelectrophoresis step 206, and described in detail below. The light wouldbe conveyed to the second PCR chamber 5706 by an optical fibre. Becausethe Peltier heater/coolers are positioned in front of and behind thesecond PCR chamber 5706, the light for the detection is introduced fromthe side of the cartridge 9. The light source may be a laser, forinstance of the type and/or with the set up discussed further below inthe electrophoresis step 206. As an alternative, however, it is possibleto use a light emitting diode based light source, as described below.

Depending upon the quantity, the number of cycles used in the PCRchamber 416 may be increased, decreased or kept at the normal level, soas to provide a quantity of DNA within the desired range after PCR hasbeen completed in PCR chamber 416.

In the context of real time quantification and/or the handling ofsamples from crime scenes (rather than those taken under controlledconditions from individuals), differences in the implementation of theinvention may be provided. These may include:

1) The parallel processing of the sample so as to allow the results froma first processing of the sample to inform on the optimum conditions etcto be used in the main processing of the sample. Further details of suchan approach are to be found in WO2009/098485, the contents of which areincorporated herein by reference with respect to the parallel processingand consideration of samples and the feedback of information from oneprocessing to the other.

2) The efficiency of the extraction should be as high as possible, forinstance through optimised sample recovery, lysis and amplification. Theuse of various processes and/or reagents to separate the DNA of interestfrom problematic components, such as PCR inhibitors, is beneficial inthis respect.

3) The cartridge used will feature many of the steps and componentsexemplified above, but with the incorporation of the parallel PCRcircuit and the ability to analyse the results therefrom, for instanceusing a laser or LED to apply light to the liquid, with the return lightbeing detected to inform on the PCR process. Photo diodes and/or camerascan be used in the light detection. A control material may be providedwithin the sample to provide a reference value with respect to the lightdetected.

4) The instrument would benefit from being able to run positive and/ornegative controls. These could be run in the same cartridge as thesample. The controls may be handled by the operator in the same manneras the sample of interest so as to inform on contamination risks. Thecontrols may just be run periodically so as to check on the instrument,for instance in the form of a calibration check.

Cartridge Components

Within the cartridge are a significant number of components, with eachbeing optimised with respect to its role and its role in combinationwith the other components.

1) Valves

To minimise manufacturing costs and give consistent operation, all ofthe valves in the cartridge are one of two types. The two types are aclosing valve 2000; FIG. 10 a; and an opening valve 2002; FIG. 10 b.

The closing valve 2000 is shown schematically in FIG. 10 a. The closingvalve 2000 is positioned above, relative to the direction of gravity,the channel 2004 to be closed. The closing valve 2000 is formed by aconduit 2006 which is in fluid communication with the channel 2004 andis in fluid communication with the bottom of a valve reservoir 2008. Thevalve reservoir 2008 is filled with paraffin wax and is 3 mm in diameterand is provided with the conduit 2006. On the top of the valve reservoir2008, a gas passage 2010 provides fluid communication with a valve gasreservoir 2012. The valve gas reservoir 2012 is full of air.

The dotted line in FIG. 10 a shows that part of the location of theclosing valve 2000 which is in contact with a heater element, not shown,provided on the adjoining printed circuit board of the instrument.

When the closing valve 2000 is to be activated, the heater element iscaused to heat up. This both melts the paraffin wax in the valvereservoir 2008 and causes the air in the valve gas reservoir 2012 toexpand. The expansion of the air provides the driving force to displacethe melted paraffin wax from the valve reservoir 2008 into the conduit2006 and then into the channel 2004.

The volume of paraffin wax displaced is controlled by the temperature towhich the valve gas reservoir 2012 is heated (variation in pressure) andthe duration of the heating applied (as the paraffin wax soon solidifiedonce the heating is switched off).

Continued displacement of the paraffin wax into the channel 2004 causesthe paraffin wax to expand in each direction along the channel 2004.

In some cases, the fluid in the channel will not compress or move in onedirection (or is limited in the extent possible) and so the flow of theparaffin wax within the channel 2004 occurs preferentially in the otherdirection. Normally, the paraffin wax is displaced into the channel 2004until a 2 mm to 10 mm length of the channel 2004 is filled. With theheat removed, the paraffin wax sets in this new position and the channel2004 is reliably sealed.

The section where the channel 2004 is to be shut, is deliberately chosento be horizontal, relative to the direction of gravity, as this assiststhe retention of the paraffin wax at the location to be sealed.

To assist further in the formation of the seal, it is beneficial toarrange the closing valve so that it is between one or two upward,relative to the direction of gravity, bends. As shown in FIG. 10 a thebend 2014 provides assistance in the accurate formation of the sealwithin the channel 2004.

The opening valve 2002 is shown schematically in FIG. 10 b. The openingvalve 2002 is positioned as a part of the channel 2004 the fluid flowsthrough. The opening valve 2004 is formed by a valve chamber 2020 whichhas an inlet 2022 from the channel 2004 in a first side wall 2024 and anoutlet 2026 leading to the continuation of the channel 2004 in theopposing side wall 2028.

The paraffin wax is positioned in the initial section 2030 of the valvechamber 2020. Downstream of this initial section 2030, is a trap section2032. The dotted line in FIG. 10 b shows that part of the opening valve2002 which is in contact with a heater element, not shown, provided onthe adjoining printed circuit board of the instrument.

When the opening valve 2002 is to be activated, the heater element iscaused to heat up. This melts the paraffin wax in the initial section2030. By the time the paraffin is melted, or shortly thereafter, anelectrochemical pump upstream of the opening valve 2002 has beenactivated for sufficient time to cause a pressure build up, upstream ofthe opening valve 2002. This pressure causes the driving force todisplace the melted paraffin wax from the initial section 2030 anddownstream into the trap section 2032. Once in the trap section 2032,the passage 2034 above the paraffin wax is clear allowing fluidcommunication through the opening valve.

With the heat removed, the paraffin wax sets in this new position andthe channel 2004 and passageway 2034 is reliably opened.

The section where the channel 2004 is to be opened is deliberatelychosen to be horizontal, relative to the direction of gravity, as thisassists the retention of the paraffin wax in the trap section 2032.

In some applications, particularly those close to the high temperaturesused in the PCR chamber, the valves benefit from using a high meltingpoint wax. This melts at greater than 95° C. and so does not melt underPCR conditions. In some cases, the valve performance can be improvedfurther by using a high melting point and lower melting point mixture;with the lower melting point wax tending to fill any cracks which formin the higher melting point wax.

A further valve embodiment is shown in FIG. 47. The channel 47-100 isconnected to the valve by a side channel 47-102 as usual. The sidechannel 47-102 leads to a first chamber 47-104. This is connected via ashort channel 47-106 to a larger second chamber 47-108.

2) Chambers

Within the cartridge, a variety of chambers are provided for a varietyof purposes. To achieve those purposes efficiently and effectively, thechamber designs are optimised in various ways.

With respect to the incubation chamber 358, this is provided with abroad base which is generally horizontal. In operation, the offsetmagnet (not shown) is used to restrain the magnetic beads in positionduring washing and during elution. The broad base provides a suitablelocation to which the beads can be drawn and secured, whilst exposingthem to the wash flow or to the elution flow.

The sloping walls within the incubation chamber 358 and the bubblemixing chamber 342 are provided to promote the flow of eluent,introduced into the chambers at the top, to the outlet at the bottom ofthe chamber.

The angular corners are used to generate improved pressure gradientsfrom the inlet for a part of the process to the outlet in thatrespective part of the process.

The first further mixing chamber 332 and second further mixing chamber336 are provided to encourage non-laminar flow within the flow route. Asthe fluid transitions from the channel, with its cross-section, to thechambers, with their increased cross-section, non-laminar flow arises.This gives good mixing for the different density fluids and particleswhich are all to be mixed. Such mixing forms are significantly better inthis respect than bubble mixing alone or piezoelectric based mixing.

The PCR chamber 416 has two principle embodiments; as described above.In each, the PCR regents are provided within the degradable shell of abead located within the PCR chamber 416. To ensure proper flow of theliquids around and past the bead, the bead is provided with a bead seat.This provides a defined rest position for the bead, but as the bead isonly contacted at discrete locations when in the seat, fluid is stillable to flow past the bead. The seat ensures that the bead does notblock at inlet to and/or outlet from the PCR chamber 416. The seatensures that there are no large areas of the bead surface, and hence ofthe reagents, which are isolated for fluid contact.

In the second of the PCR chamber 416 embodiments, described in thealternatives for the cartridge section, the PCR chamber 416 iscompletely filled with fluid. This gives a reproducible volume of fluidin the PCR process. The same position arises with the third embodiment,FIG. 22.

In the first of the PCR chamber 416 embodiments, the maximum level offluid within the PCR chamber 416 is controlled by the relative height ofthe outlet within the chamber. The outlet in effect acts as an overflowfor the fluid, once the PCR chamber 416 has filled to this level. A headspace remains above the fluid, within the PCR chamber 416.

3) Vents

To allow fluid flow, air or sample, around the cartridge 9, variousvents need to be provided for various chambers.

To prevent any risk or suggestion that material can enter the cartridge9 through such vents, each of the vents is provided with a filterelement to exclude particulate material. In addition, when a vent ispart of the active processing on the cartridge 9, the vent is underpositive pressure and so air is flowing out through the vent. This tooassists in preventing any risk of particulate material entering thecartridge 9.

In some situations, it is desirable to be able to allow air to passthrough the vent freely, but for the vent to resist the passage of anysubsequent liquid. An example is to be found in the alternative PCRchamber 416 filling embodiment. To provide this, those vents arehydrophobic. The vent may be hydrophobic because of the base materialforming the vent and/or because of a treatment applied to the materialof the vent. Such a treatment can be provided, for instance, by usingpolypropylene material and/or by providing a polysulphone coating.

4) Archive

As described above, the fluid not needed in the PCR chamber 416, ispumped onward to an archive chamber 422.

The purpose of the archive chamber 422 is to provide a storable recordof the sample supplied to the sample amplification stage 204, and thePCR chamber 416 in particular.

If needed, the sample in the archive chamber 422 can be accessed at alater date to enable a further amplification and analysis to beperformed. Further processing in this way is useful where it isnecessary to repeat the analysis, for instance by way of verification.Alternatively, further processing enables a different amplification andanalysis protocol to be applied, for instance, a protocol suitable forlow levels of DNA within the sample.

In the form shown in FIG. 3, the archive chamber 422 is an integral partof the overall cartridge 9.

In an alternative, form shown in FIG. 11, the archive chamber 2422 isstill fed the surplus sample through a channel 2418 leading away fromthe PCR chamber, not shown.

The archive chamber 2422 is positioned on a stub 2750 which extends fromthe side of the cartridge 9. The stub 2750 is connected to the cartridge9 during normal use, but a line of weakness 2752 is provided. Thisallows the stub to be snapped off the cartridge 9 after the completionof the processing. This means the archive function can be provided byonly storing the stub 2750, rather than have to store the far largeroverall cartridge 9. Given the number of samples which may beconsidered, and the time for which they have to be stored, saving ofstorage space is a significant issue.

To seal the archive chamber 2422, once it has been loaded, a closingvalve 2754 is provided on the cartridge 9 side of the line of weakness2752 and a further closing valve 2756 is provided on the stub 2750 sideof the line of weakness 2752. These valves are activated to placeparaffin wax in the channel 2418 on either side of the line of weakness2752. To provide for long term storage, a further closing valve 2758 isprovided on the channel leading from the archive chamber 2422 to thevent 2424.

Just as the cartridge 9 is provided with an identifier, which is used tolink it in the records to the sample loaded upon it, then the stub 2750is also provided with a common identifier so as to maintain the linkafter the stub 2750 is broken off the cartridge 9.

5) Reagents

Various options exist for the provision of the reagents needed in thevarious steps of the processing. As far as possible, so as to keep theprocessing as simple as possible for the user, the cartridge 9 isprovided with pre-loaded reagents. Examples of such pre-loaded reagentswould include the bead provided in the PCR chamber 416; with the beadcarrying the PCR regents inside. Other pre-loaded regents include thevarious wash liquids and elution liquids described in the methodologyabove.

If necessary, one or more reagents can be provided separate from thecartridge 9, and be loaded onto the cartridge at or close to the time ofuse. This may be necessary where the reagent is unable to withstandprolonged storage under the conditions to which the cartridge 9 isexposed. These may be conditions of temperature and/or mechanicalconditions such as vibration or orientation.

A preferred form of reagent provision is provided where the reagent(s)are provided as part of a solid phase reagent or solid phase reagentstorage component, with release of the reagent being triggered by anincreased temperature. Gel forms of reagent and/or reagent storagecomponent, preferably triggered to release by the application of highertemperatures are also a useful option.

6) Electrochemical Pumps

To simplify the construction and costs of the cartridge, a commonapproach is used to providing the motive power to the various operationson the cartridge; electrochemical pumps. Each of the electrochemicalpumps consists of a pair of electrodes immersed in the electrolyte. Theflow of a current results in off gassing. The off gas collects in thetop of the electrochemical pump, increases in pressure and leaves thepump via the outlet in the top of the pump. This off gas pushes ahead ofitself other fluids encountered in the channels and chambers. The offgas contributes to bubble mixing in some of the stages.

To give a desired extent of pumping, the volume of the electrochemicalpump can be varied. The extent of pumping can be delivered in one, twoor more goes, as turning off the current stops the pumping action.

The rate of pumping and/or pressure delivered can be varied by varyingthe molarity of the electrolyte. Sodium chloride is the preferredelectrolyte; used at 1M; and used in conjunction with aluminiumelectrodes.

7) Electrophoresis Matrix

The material provided within the capillary of the electrophoresis stageis important to the reliability and resolution of the analysis obtained.

Various possible materials can be used in the capillary. These includethe use of polymer matrix, for instance a polyhydroacrylamide, apolydimethylacrylamide or mixtures there of. The polymers may becross-linked to give the desired properties and/or formed into theirstate of use within the capillary, after loading. It is also possible touse an inert bed of particulate material to form the matrix in which thesize based separation is achieved.

As well as optimising the performance through the properties of the gel,it is also possible to treat the capillary walls to improve properties.For instance it is possible to apply hydrophilic coatings, such aspoly(hydroxyethlacrylamide).

A potential methodology for the electrophoresis matrix is to store thatmaterial in a chamber which is a part of the CE chip, but not use thatchamber for the CE separation. Instead, when required for use, thestored matrix is moved from the chamber into the capillary so as to fillit to the desired degree. As a result of loading just before use, thematrix is no subject to sedimentation effects; these can have adetrimental effect on the analysis. Pressure loading can be used forthis purpose.

Another potential methodology is to fill the main channel and arms ofthe CE chip with the matrix. Those parts of the CE chip where the matrixis not needed, for instance aside from the main channel, may be masked.In this way, when UV light is applied the parts where the matrix is notneeded retain the matrix unaltered. The unaltered matrix can be washedaway. Where the matrix is exposed to UV light it is altered and resistswashing away.

8) CE Chip Design

A preferred configuration for the CE chip is shown in FIG. 42 a and thedetailed partial view of FIG. 42 b.

The end portions 42-100 cooperate with the carrier when the chip ismounted within it. The external profile of the base of the CE chip isdesigned to match with that defined by the raised surface around the CEchip heater board, described elsewhere in this document.

As described below, a number of electrodes are required in differentparts of the channels provided within the CE chip so as to load thesample and then perform the necessary separation to give the analysis.These electrodes within the channels are connected to pins 42-102 whichextend above the plane of the CE chip. These pins 42-102 are positionedso that they are within the cut away portion of the second support andso are exposed. This allows suitable connections to be made to thesepins 42-102 so as to apply the necessary voltages to them and to theelectrodes connected to them.

The CE chip is shown with a single channel in which CE is performed, butchannels suitable to perform separations on multiple samples could beprovided.

9) PCR Chamber Sealing

In the embodiments described elsewhere, the chambers and the valveswhich are used to seal the channels leading to and from them areseparate. In the following embodiment, the chambers and the valves areintegrated as a single component.

As shown in FIG. 41 a, the PCR chamber 41-100 is provided in thecartridge. However, the walls defining the circumference, at least, ofthe chamber 41-100 are rotatable within the body of material forming thecartridge. In the lefthand form, the rotatable wall is positioned suchthat the holes therein are aligned with the inlet channel 41-102 and theloading outlet channel 41-104. As a result, liquid can enter and gasleaves the chamber 41-100 until the chamber is full, centre form. Therotatable wall can then be rotated to align the holes therein with theinlet channel 41-102 and the dispense outlet 41-106, right hand form, toallow the contents to be emptied.

A variant of this approach is shown in FIG. 41 b, where inlet channel41-100 is connected to outlet channel 41-108. Rotation aligns the holeswith dispense inlet 41-110 and dispense outlet 41-106.

The variant in FIG. 41 c uses the arrangement to seal the chamber duringPCR. In the left hand form, the inlet channel 41-102 is connected to andfills the chamber up to the level of the outlet channel 41-108. Partialrotation offsets the holes in the rotating wall from alignment with anyof the inlets/outlets, centre form. After PCR, further rotation alignsthe holes with the dispense inlet 41-110 and dispense outlet 41-106.

The extent of rotation may be limited by abutment surfaces provide inthe cartridge wall which abut surfaces on the rotating walls or viceversa. Partially circular forms for the hole in the cartridge whichreceives the rotating walls and/or vice versa may also be used tocontrol or limit rotation in one or both directions.

Rotation may be provided by cooperation between an actuator and a slotin the circular wall.

Rotation may cause pads or other pliable material to be compressed orotherwise deformed to give sealing.

One or more of the channels may serve as a light path, rather than or inaddition to being a fluid flowpath, so as to allow an investigatoryinstrument to shine light into the liquid contained within the chamber.Such an embodiment is useful in the context of the cartridge variant forreal time PCR discussed above.

Instrument Configuration and Appearance

The instrument 11 is illustrated in FIG. 12 and is provided within acasing 8000. The mid section 8002 of the instrument 11 is provided witha door 8004 provided with a latch 8006. Behind the door 8004 is thelocation at which the cartridge 9 is mounted in use. This location is aposition in which the plane of the cartridge 9 is parallel to the planeof a printed circuit board 8008. At the location, the cartridge 9 andcomponents on the printed circuit board 8008 contact one another.

Behind the printed circuit board 8008 are the electronics for operatingand controlling the components provided on the printed circuit board8008. These include the power supplies, voltage controllers, temperaturecontrollers and the like.

The upper section 8010 of the instrument 11 provides the display 8012 bymeans of which the user inputs information into the instrument 11 andreceives visual information from the instrument. The software andhardware for operation of the display 8012 are provided on a computerpositioned behind the display screen 8012 in the upper section 8010.

The lower section 8014 of the instrument 11 contains the high voltagepower supply and controller for the laser used in the inspection of thecapillary electrophoresis. Also in this lower section 8014 are thecharge couple device used to sensor the fluorescence and the optics forconveying the light to and from the capillary.

Another embodiment of the instrument is shown in FIGS. 29 a, 29 b and 29c. The instrument 29-11 is provided within a casing 29-8000. The uppersection 29-8002 of the instrument 11 is provided with a door 29-8004.The door 29-8004 is a combination of a top section 29-8006 and frontsection 29-8008 of the casing 29-8000.

The lower section 29-8010 of the instrument 11 provides the display29-8012 by means of which the user inputs information into theinstrument 11 and receives visual information from the instrument 11.

The window 29-8014 allows for visual inspection of the cartridge used. Aseries of light bars 29-8016 are used to indicate the extent of progressthrough the steps involved; the more of the bar which is lit the greaterthe extent of the step performed.

A stylus 29-8018 is used by the operator to interact with the display29-8012.

Various control buttons 29-8020 are provided below the screen 29-8012.

The overall dimensions of the instrument are width, W, 419 mm, overallheight, OH, 621 mm, depth, D, 405 mm.

The side panel 29-8022 is removable for maintenance purposes.

The embodiment of FIG. 30 shows the door 30-8004 structure more clearly,together with the workspace 30-8024 that is accessed through it. Theworkspace 30-8024 includes the slot into which the cartridge carrier30-8026 is inserted. The cartridge carrier 30-8026 is as describedelsewhere in this document. The workspace 30-8024 also includes the lanefinding apparatus 30-8028.

The cover 30-8030 in the side panel 30-8032 is opened by rotation toallow access to the optics for maintenance purposes.

Cartridge to Instrument Interface

As described above, once the cartridge 9 is loaded with the sample, thecartridge 9 is loaded into the instrument 11 for the processing to beconducted.

As a first step, the latch 8004 is released and the door 8002 is opened.

To insert the cartridge 9, FIG. 13, the section of the cartridge 9 whichbears the PCR chamber 416 is inserted into a slot 8023 between thecomponents which will control the PCR process. These components includethe thermoelectric heaters/coolers, Peltier devices 8025, and fans 8027there for. These components are free to travel to a limited extent tohelp with the locating of the cartridge 9 within the slot 8023, whilstbeing forcibly returned to the optimum position after insertion so as togive effective heating/cooling.

The cartridge 9 is provided with a series of recesses which cooperatewith dowels extending through the printed circuit board 8008 toaccurately register the cartridge 9 relative to the printed circuitboard 8008. The dowel arrangement is such that the cartridge 9 cannot befitted the wrong way round.

Once positioned, the cartridge 9 is provided in a plane which isparallel to the plane of the printed circuit board 8008. Both componentshave flat surfaces facing one another so as to assist with the goodcontact needed between them.

The closing of the door 8002 and operation of the latch 8004 applies acompressive force to the cartridge 9 by way of a series of spring loadedpins mounted on the inside surface of the door 8002. This helps hold thecartridge 9 in firm contact with the printed circuit board 8008.

The printed circuit board 8008 is important to the successful operationof the invention. It provides the energy sources for the variouscomponents to be driven on the cartridge 9. In effect, the drivers areall provided in the cartridge 9, but the energy sources are provided onthe printed circuit board 8008. In this way, the precision operationneeded is ensured by the expensive and bespoke electronics andarrangement of the printed circuit board 8008; a reusable component ofthe instrument. In this way, the cartridge 9 is simple andself-contained. This reduces the complexity of the interface between thetwo and also removes the risk of contamination of the contents of thecartridge 9. The only transfer between the printed circuit board 8008and the cartridge 9 is conducted and radiated heat from the heaters andthe magnetic field provided by the magnet.

The components provided on the printed circuit board include:

-   -   d) The electrical contacts 9000 which connect to the pins of the        electrochemical pump electrodes on the cartridge 9. These        provide the electrical power, when needed, to operate the        electrochemical pumps.    -   e) The electrical heaters 9002 which are used to apply heat to        the valves on the cartridge so as to open or close the valves        depending upon their type. These are square areas of resistance        heating material which is applied by printing a paste to the        desired location. The heating effect is improved if the square        block is rotated through 45° relative to the axis of the channel        subject to the valve.    -   f) The magnet 9004 which is advanced into proximity with the        cartridge 9 when it is desired to retain the beads and prevent        them from moving. The magnet 9004 is retracted away from the        cartridge 9 when it is desired to release the beads within the        chamber 358.    -   g) The sensors 9006 are providing feed back and/or verification        of the conditions induced by the heaters etc.

Alternatives for Cartridge to Instrument Interface

If it is necessary to alter or improve the contact between the cartridgeand the printed circuit board, there are various options for doing so,including the following:

-   -   a) The loading provided by the sprung pins mounted on the door        8002 can be increased. This applies a force to the cartridge 9        and pushes it against the printed circuit board 8008.    -   b) The cartridge 9 can be mechanically clipped to the printed        circuit board 8008, with the clip(s) applying a compressive        force.    -   c) The cartridge 9 can be provided with a compressible substrate        mounted on the surface which is intended to contact the printed        circuit board. In this way, when then cartridge 9 and printed        circuit board 8008 are pushed together, the substrate will        provide good all over contact. The substrate can be a solid        material, paste or even a liquid. The materials of the        substrate, or parts there of, are selected so as to provide        maximum thermal conductivity, for instance. Particles,        nanoparticles or other materials may be added to alter the        properties. The substrate may be protected, prior to use, by a        peelable backing.    -   d) As described above, the components (such as heaters etc) are        provided in a fixed position on the printed circuit board 8008.        This means they move with the printed circuit board 8008. It is        possible to provide one or more, and even each of these        components with a degree of independent movement. For instance,        they may be provided with a sprung mounting on the printed        circuit board. In this way, each is able to independently adjust        its position, forward and backwards, relative to the cartridge.    -   e) As shown in FIG. 23, it is possible to provide the section of        the cartridge 9 which bears the PCR chamber 416 in opposition to        stacked components which will control the PCR process. In this        example, the stack includes a first Peltier device 23-01 in        contact with the cartridge 9 and in contact with and aligned        with a second Peltier device 23-03. The stacking of the devices        allows high temperatures, for instance greater than 150° C. to        be obtained within the PCR chamber. Such temperatures are        beneficial in terms of melting the high melting point wax seals        described elsewhere within this document.    -   f) Alternative forms of heater may be used instead of Peltier        effect device. For instance infra red heating devices may be        used. The material around the PCR chamber, or a part of that        material, may be capable of resistance heating to give the        necessary heating for the chamber. Resistance heaters positioned        against the cartridge may be used. Microwave heating may be        used.

Alternative Cartridge to Instrument Interface

In the alternative embodiments of the instrument described above inrelation to FIGS. 29 a, b, c and FIG. 30, the cartridge is not loadeddirectly into the instrument. Instead, once loaded with the sample, thecartridge 31-01 is loaded into a cartridge carrier 31-03.

The use of the carrier 31-03 means that the cartridge 31-01 and the CEchip can be constructed separately. This allows different materialand/or different production tolerances to be used for the differentcomponents; a beneficial effect on cost and/or performance and/or thebalance between those can thus be provided.

The carrier 31-03 also allows for easy assembly of the requiredcomponents and their insertion into the instrument in a unitary form. Atthe same time, the carrier is designed so as to allow separate alignmentchecking and adjustment for the cartridge and the CE chip so that bothare in their correct, optimised position within the instrument.

If desired, the cartridge position can be checked and any alignmentadjustment necessary can be made. Before CE starts, a separate check canbe made on the alignment of the CE chip, within any adjustments it needsbeing made before CE starts.

The cartridge carrier 31-03 is illustrated in FIG. 31 a. The cartridgecarrier 31-03 includes a first support 31-05 and a second support 31-07which is perpendicular to the first support 31-05.

The first support 31-05 is used to carry the cartridge 31-01. The secondsupport 31-07 is used to carry the capillary electrophoresis, CE, chip;this interaction is described further below.

The prepared cartridge 31-01 is presented with its face 31-09 to theface 31-11 defined by the first support 31-05. An externally threadedscrew 31-13 provided at each corner of the first support 31-05 isreceived into an opposing aperture 31-15 provided at each corner of thecartridge 31-01. Rotation of the screws 31-13 causes them to engage withand enter an internal screw thread provided in the apertures 31-15.Further tightening mounts the cartridge 31-01 on the first support 31-05and hence the carrier 31-03 in a secure and known position.

The interaction between the cartridge 31-01 and the carrier 31-03 isshown in more detail in FIG. 31 b in relation to one of the screws31-13.

The screw 31-13 is provided with a knurled head 31-17. The threadedengagement occurs between the end 31-19 of the screw 31-13 and theaperture 31-21 in the cartridge 31-01. A jam nut 31-23 in cooperationwith a washer 31-25 serves to hold the screw 31-13 on the carrier whennot engaged with a cartridge 31-01 The jam nut 31-23. washer 31-25 andsleeve 31-27 serve to prevent over tightening between the carrier 31-03and the cartridge 31-01.

Rotation of the screw 31-13 pulls the knurled head 31-17 and thecartridge 31-01 closer together. This causes compression of the conicalspring 31-29 between the knurled head 31-17 and an abutment surface31-31 on the first support 31-05. The spring 31-29 assists in ensuringcorrect alignment during tightening. Once rotation is finished, thefirst support 31-05 and hence carrier 31-03 is in a known positionrelative to the cartridge 31-01.

The CE chip 32-31 is inserted into the carrier 32-03 as shown in FIG. 32a. The CE chip 32-31 is slid into a slot. As shown in FIG. 32 b, thesecond support 32-07 provides such a slot 32-33 at either end forreceiving the end portions 32-35 of the CE chip 32-31. An incline 32-37on the lead edge 32-39 of the CE chip 32-31 engages with the end 32-41of a spring loaded plunger 32-43 and causes it to displace outward,arrow A. Once the recess 32-43 is presented to the end 32-41 of theplunger 32-43, the plunger 32-43 returns, arrow B, and so preventsonward movement of the CE chip 32-31 past the desired position.

Once the cartridge 31-01 and the CE chip 32-31 are inserted into thecarrier 31-03, 32-03, the fluid connection between the two is providedby a tube 33-45. The insertion of the cartridge 31-01 into the carrier31-01 causes the electrophoresis step inlet 28-570 on the cartridge31-03 (see FIG. 28 a) to become connected to the tube 33-45. As shown inFIG. 33 a, the tube 33-45 extends upward, parallel to the plane of thecartridge 31-01 and the first support 31-05 through an opening 33-47 inthe carrier 31-03. As shown in FIG. 33 b, once through the opening33-47, the tube 33-45 makes a 90° turn into the plane of the secondsupport 31-07 and the CE chip 32-31. The tube 33-45 is accommodatedwithin the second support 31-07 above the CE chip 32-31. A further 90°turn leads the tube 33-45 into the CE chip 32-31. The remaining fluidtransport is handled within the CE chip 32-31 itself, as describedelsewhere in this document.

After insertion of the cartridge 31-01 and the CE chip 32-31 into thecarrier 31-03, as described above, the carrier 31-03 is ready forinsertion.

As a first step, the door 34-8004 is opened, FIG. 34 a, to expose theworkspace 34-8024. The work space 34-8024 includes the slot 34-47 thatthe carrier 34-03 is inserted into.

The carrier 34-03 is inserted into the slot 34-47 until the secondsupport 34-07 comes to rest on the surface 34-49 of the workspace34-8024. The cooperation of the carrier 34-03 with the slot 34-47ensures the correct general positioning of the cartridge 34-01 withrespect to the instrument, both in terms of lateral and verticalpositioning; FIG. 34 b.

Insertion in this way provides the section of the cartridge which bearsthe PCR chamber between the components which will control the PCRprocess; as described further below.

Once inserted, the door 34-8004 is closed. The closing of the door34-8004 triggers various actions based upon contact between the closeddoor 34-8004 and casing. The clamping of the cartridge to the PCB, thepositioning of the CE chip on the CE chip heater board, the introductionof the electrical contacts to the pins provided on the CE chip, theintroduction of the electrical contacts to the pins providing theconduction path to the electrodes in the electrochemical pumps are alltriggered in this way. The closure of the door 34-8004 is also used toturnoff the interlock for various safety systems within the instrument.The interlock prevents, for instance, the laser being active with thedoor or any other opening in the instrument's casing being open. asimilar principle applies to the power supplies within the instrument.

As with other embodiments, it is important to provide effective andaccurate contact between the cartridge and the instrument interface. InFIGS. 35 a, b and c the provision of the contact is illustrated.

FIG. 35 a shows the carrier 35-03 in position in the slot 35-47. In theinsertion position, as shown, the arrangement provides for a gap 35-51between the face 35-53 of the cartridge 35-01 which opposes the face35-55 of the printed circuit board 35-57 of the instrument.

In the next step, FIG. 35 b, the cartridge 35-01 is moved into the useposition. A platen 35-59 is moved, direction of arrows, by an actuator,not shown. This causes the cartridge 35-01 to be brought into fullcontact with the PCB 35-57. The movement is such that the conical spring35-29 is further compressed. During this movement, a series of rodswhich extend through the PCB 35-37 enter various holes (27-13 in FIG.27) and so ensure that the alignment between the cartridge and the PCBis correct in that orientation too.

When the use of the cartridge 35-01 has finished, then the force appliedto the platen 35-59 by the actuator is released. As a result, thecarrier 35-03 is returned to the insertion position by return springs,not shown. The release causes the conical springs 35-29 to pull thecartridge 35-01 back into position inside the carrier 35-03, FIG. 35 c.The carrier 35-03 can then be removed by lifting it out of the slot35-47, taking with it the cartridge 35-01.

The face to face contact between the cartridge and the PCB provides themajority of the interactions between the cartridge and the instrument,for instance, heating for valve control, sensor etc. The contact betweenthe PCR chamber and its temperature cyclers are provided through furthercomponents, however; see FIGS. 36 a, b, c and d.

In FIG. 36 a, the cartridge 36-01 is shown inserted into the slotprovided in the instrument. Once inserted, the section of the cartridge36-01 bearing the PCR chamber is positioned between a pair of calipers36-100. The PCB is cut away at this location so as to not be in the wayof the Peltier effect devices 36-102, 36-108 and pair of calipers36-100. The calipers 36-100 are floating such that they do no interferewith the contact sought between the cartridge 36-01 and the PCB duringthe movement from the insertion position to the use position.

The front caliper 36-100 a is provided with a Peltier effect device36-102 mounted on a support 36-104 which is capable of reciprocatingmovement, arrow C, under the control of actuator 36-106. The actuator36-106 is also mounted on the pair of calipers 36-100.

The back caliper 36-100 b is provided with a second Peltier effectdevice 36-108 mounted fixedly on the caliper 36-100 b. The secondPeltier effect device 36-108 is provided in opposition to the Peltiereffect device 36-102.

In the open position shown in FIG. 36 c, such as is provided with thecartridge in the insertion position, the distance between the opposingfaces 36-110, 36-112 of the Peltier effect device 36-102 and the secondPeltier effect device 36-108 is more than the thickness of that sectionof the cartridge 36-01 and more than the thickness of the carrier 36-03which passes between the pair of calipers 36-100 during insertion of thecarrier 36-03.

In the closed position shown in FIG. 36 d, such as is provided duringthe amplification step, the distance is reduced. This is achieved by theactuator 36-106 moving the Peltier effect device 36-102 on the frontcaliper 36-100 a towards the cartridge 36-01 and towards the opposingsecond Peltier effect device 36-100 b. This actuation, combined with thefloating nature of the pair of calipers 36-100 brings both of thePeltier effect devices into firm contact with the cartridge 36-01 onopposing sides thereof. They are now in position to provide thenecessary heating and/or cooling for the PCR step.

Thermocouples to sense the temperatures applied, and potentially to beused to control the temperatures applied, are provided in closeproximity with the Peltier effect devices, embedded in copper shims,bonded to the Peltier effect devices.

Before the carrier 36-03 is removed, the actuator 36-106 returns thePeltier effect devices 36-100 to the open position.

In addition to the carrier allowing for relative movement of thecartridge to ensure correct positioning with respect to the PCB, thecarrier also allows for totally independent relative movement of the CEchip. This is importing in ensuring correct positioning of the CE chipfor the CE step. This is achieved by the structure and operation shownin FIGS. 37 a and b.

As the carrier 37-03 with the CE chip 37-31 in it is inserted into theslot in the instrument, the second support 37-07 approaches the worksurface 37-49. The work surface 37-49 carries a CE chip board heater37-100 in the form of a planar surface. this is surrounded by a raisedsurface 37-102 which provides a nest for the CE chip 37-31 oncepositioned.

Projecting pins 37-104 on the work surface 37-49 enter apertures 37-106provided in the second support 37-07 of the carrier 37-03; FIG. 37 a. InFIG. 37 b, the top part of the second support 37-07 is shown cut away sothat the full extent of the CE chip 37-31 can be seen. The apertures37-106 in the second support 37-07 align with the slot 37-108 whichreceives the end portions 37-108, 37-110 of the CE chip 37-31. As aresult, the end portions 37-108, 37-110 are also provided with throughapertures 37-112 a, 37-112 b. The projecting pins 37-104 thus passthrough these apertures 36-112 a, 36-112 b too as the carrier 37-03approaches the work surface 37-49.

The conical ends of the pins 37-104 mean that they enter the apertures37-106, 37-112 a, b, even where there is potential misalignment. Thefuller diameter parts of the pins 37-104 encourage the CE chip 37-31into the correct position. The CE chip 37-31 is centred to the CE chipboard heater 37-100 as a result. The CE chip heater board 37-100 andraised surface 37-102 can be seen clearly in FIG. 38.

Electrophoresis Components 1) Optics

In the electrophoresis step 206, at the detection location 628, lightfrom a laser 800 is focussed to be incident upon the fluorescent dyeassociated with a DNA element to make it detectable.

A different dye is used for each different DNA element type; a type isgenerally associated with a given locus.

To get good sensitivity, it is important for the incident light to be ofsufficient intensity for the detectors to receive sufficient light to besensitive to the emitted fluorescent light, but for the intensity not tobe so high as to give rise to photobleaching of the dyes. To provide forthis, the following arrangement is used; FIG. 14.

The light source is a compact laser 900 which is mounted on a heat sink902. The laser 900 is a Cobolt Calypso laser (from Cobolt AB, Kraftriken8, SE-104 05, Stockholm, Sweden) and emits at 491 nm with a maximumpower of 50 mW. The light emitted by the laser 900 is fed to a fibrecoupler 904 (09 LFC 001, f=3.5 mm from Melles Griot, 2051 PalomarAirport Road, 200, Carlsbad, Calif. 92011, USA) and hence into an patchcable assembly (M31L01, from Thorlabs, 435 Route 206 North, Newton,N.J., 07860, USA) and optical fibre 906 (GIF625, dia 62.5:m, NA=0.275from Thorlabs, 435 Route 206 North, Newton, N.J., 07860, USA).

The use of the optical fibre 906 is beneficial as it safely controls thelaser light direction, enables the laser light to be easily conveyed tothe position of use and enables mechanical stability to be providedwithin the overall system. At the end of the optical fibre 906 a powerof up to 45.32 mW is still observed.

The laser light then passes through a collimator 908 (F230FC-A, F=4.5mm, NA=0.55, from Thorlabs) and a logpass filter with a sharp cut-offwavelength, EM filter (Omega Optical XF3093, T50=515 nm) before reachingthe spot mirror 910.

The spot mirror 910 is used to both direct the laser light to thedetection location 628 of the capillary and to transmit, anisotropicallyand without filtering, the fluorescent light received there from to thedetector unit. It is angled at 45° to the beam of laser light. To dothis, the reflector 910 consists of a 25 mm round glass disc whichtransmits all light from <80 above 380 nm. An ellipse, 2 mm long by 1 mmwide, is provided at the centre of the reflector 910 (so as to presentan effective 1 mm circular mirror), formed of a highly reflective mirrorlayer deposited there (reflectivity of 99.99%).

Before reaching the detection location 628, the laser light passesthrough a focussing lens 912. This can be a microscope optic or othersuch adjustable focussing lens. Such optics are useful as they introduceno optical aberrations to the light, shape the beam for application tothe detection location 628 and don't give any selective loss of lightcolours. The power reaching the detection location 628 is over 27.40 mW.

The fluorescent light is effectively scattered from the dye in thecapillary 616 in all directions. For the fluorescence light to reach thedetector unit, that light needs to hit the spot mirror 910 at a locationoutside of the glass spot. If it does so, the light is transmitted intothe detector unit 914.

The detector unit 914 includes a slit in front of a spectrometer toobtain diffraction-limited incident light, the spectrometer providedwith a diffraction grating and a lens 918 (LA1608A plano convex, f=50mm, D=25 mm, with anti-reflective coating within 350-650 nm, made of BK7glass, Thorlabs Inc), to direct the light to the charge coupled device916. The CCD 916 has spectroscopic abilities.

The CCD 916 generates the signals which are then used to generate theelectropherogram, an example of which is shown in FIG. 15

Using such an approach, a sensitivity approaching that of laboratorystyle electrophoresis instruments can be reached. The instrument is ableto detect down to the presence of 2.5 pM of fluorescein dye at pH 7.

In an alternative approach, certain problems with the stability of thefibre optics can be avoided by providing an open beam approach todelivering the light from the laser to the channel.

An alternative embodiment of the optics is shown in the cut awayperspective view of FIG. 39. The instrument casing 39-01 providesvarious mounts for the optics. The light is generated by the laser head39-03 operated under control by the laser controller 39-05. The lightenters the optics 39-07 and is directed at the channel in the CE chip,not shown, mounted in the CE chip heater board 39-09.

The return light enters the optics 39-07 and is directed back to thespectrometer 39-11 and CCD camera 39-13. Above the CE chip heater board39-09 is the chip alignment structure 39-15 which is described furtherbelow.

2) Calibration and Verification for Optics

When first using the optics for detecting the electrophoresis results,and periodically thereafter, it is beneficial to ensure that the opticsare properly calibrated to the capillary 616 at the detection location628 in the electrophoresis cartridge section. This ensures besttransmission of the excitation light into the detection location 628,best recovery of the fluorescence light from the dyes encountered at thedetection location 628 and the performance of the detection at thedetection location 628 (and hence at the correct distance from the pointat which the sample is injected).

To achieve these aims, the electrophoresis cartridge section is providedwith various aids. These are intended to allow automated verificationand calibration of the position by the instrument 11.

Firstly, a fixed marker is provided on the electrophoresis cartridgesection, a known distance along the capillary 616 and a known distanceperpendicular to the capillary 616, from the detection location 628.When the laser light is incident upon the fixed marker, a response isdetected by the CCD 916. The position of the incident laser light isthus known. The incident position of the laser light along the capillaryis thus correct. The known distance of the fixed marker from thedetection location 628, perpendicular to the capillary 616 can then beused to adjust the position at which the laser light is incident so asto correspond with the detection location 628. X and Y axis verificationof the incident laser light position corresponding with the detectionlocation 628 is thus provided. The marker could be a physical mark (forinstance etched) on the cartridge and/or a coloured mark (for instance adye) and/or a quantum dot.

To provide for the verification on the Z axis, the working distancebetween the lens and the capillary 616, a known source, with a knowncharacteristic is provided on the electrophoresis cartridge section at aknown Z axis distance relative to the correct Z axis distance of thecapillary 616. By adjusting the focus of the lens so as to maximise theresponse by the CCD 916, the correct working distance for the knownsource is established. An adjustment can then be made to reflect therelative working distance for the known source relative to the capillary616. Ideally, these are in the same plane at the same working distanceso as to allow the known to provide direct verification for the Z axisposition relative to the capillary 616.

As an alternative means of verification on the position, it is possibleto use the marker for the X axis and then use variation in transmissionto check the Y axis position. Thus a marker is used to determine thecorrection position along the axis of the capillary 616. The adjustmentcan then scan in the Y axial direction are use the CCD (or anotherdetector) to consider the variation with position. The reflected signalwill be constant at a level when the laser light is incident on thecartridge away from the capillary. When incident light traverses thecapillary 616, then the signal will vary in a predictable manner, soallowing the position to be set subsequently at the positioncorresponding to the middle of the capillary 616 in the signal. Toassist in this, it is possible to introduce a polariser insert for thecalibration part of the process so as to increase the observed variationin the signal. The polariser is removed before the actualelectrophoresis results collection starts. The effect whose variation isdetected can arise from the capillary 616 itself, a marker at a knowndistance from the capillary 616 or a material present in the capillary616 (for instance, a dye labelled component provided as part of a sizingstandard, whose mobility is higher than the other elements of the sizestandard or unknown elements).

The FIG. 39 and FIGS. 40 a, b and c embodiment shows the alignmentstructure 39-15 and its operation.

The alignment structure 39-15 is in the form of a swing arm 40-100 whichcan be pivoted relative to the casing 40-102 under the power of anactuator contained within the swing arm 40-100. The other end of theswing arm 40-100 is provided with a camera 40-104.

In the stowed position, FIG. 40 b, the swing arm is positioned incontact with a hard stop 40-106 mounted on the casing 40-102 too. In thecheck position, FIG. 40 c, the actuator has caused the swing arm 40-100to swing away from the casing 40-102 and so position the camera 40-106over the channel 40-108 in the CE chip 40-31.

In the use position, triggered by the operator, a laser is activated andthis creates a diffraction pattern which can be seen on the cameradisplay. The adjustment for the CE chip position is used to move the CEchip until the diffraction pattern indicates that the middle of thechannel has been located. The alignment of the channel with the opticsused in the analysis is thus provided. The camera can also be used toachieve focussing of the system in the Z axis adjustment.

3) Electrophoresis Environment Control

For the necessary resolution to be obtained in the electrophoresis step206, the temperature of the capillary 616 and its contents need to becarefully controlled at the optimum temperature. In the presentembodiment, the electrophoresis cartridge section is in contact with athermally conductive block, with a series of resistance heaters providedon the opposing side of the block. These are provided with controllersand are capable of maintaining the temperature of the electrophoresiscartridge section at the optimum temperature +/−0.3° C.

In addition, the cavity that the electrophoresis cartridge section isprovided in is thermostatically controlled at the optimum temperature.This reduces still further temperature variation before, during andafter use.

The use of a CE chip heating bed, and raised surface around it, isbeneficial in controlling the temperature within the CE chip. The nestso formed ensures consistent positioning and good contact.

4) Use of LED's as Light Source

FIG. 16 depicts a schematic of an example of a system for detectingfluorescence. The system includes light emitting diodes (LEDs), e.g.,high power cyan LEDs, to provide excitation wavelength light to detectdyes combined with biological samples. The system also includes abifurcated optical fibre assembly made, e.g., from high transmissionfused-silica cores with high numerical apertures (NAs), e.g., NA=0.22.The LED excitation system described herein can be applied for DNAdetection in capillary electrophoresis systems in mobile analyticalunits. The compactness and light weight of the LED system enablesautomating assays for nucleic acid studies. Using the compact and lightweight system allows creating bench-top analysis systems that can beused both in the laboratory and in the field.

In some implementations, two LEDs are assembled in parallel and suppliedwith a stabilized DC voltage of 3.6 V. The current passing through theLED assembly is 1.8 A. The junction is maintained at 15±1° C. by aProportional-Integrative-Derivative (PID) control loop (Model TE-36-25from T.E. Technology, Inc.) acting on two 13×13 mm thermoelectricmodules. To save power, and space, two Peltiers modules are controlledin parallel and the thermocouple sensor is placed on only one of themassuming that, by construction symmetry, they both behave similarly. Analuminum heat sink and a fan (12 V DC) complete the cooling module. Thismodule extends the lifetime of the LEDs by two orders of magnitude.Without cooling the junction, the supplied current is 2.7 A.

The first step of collimation is the use of an acrylic-molded lens fromLumiled, which collimates the emitted light to a 15° cone half-angle(NA˜n sin(2_(1/2))˜0.26). The light is then focused onto a plano-convexlens (f=35 mm, D=25 mm; NA˜D/2 f˜0.36). NA_(LED)<NA_(lens) or thenumerical apertures are matched. The distance between the apex of thelens and the plane of the collimator, L_(max), is adjusted by amicrometer screw to maximize the power read by a calibrated siliconphotodiode sensor. The value obtained (25 mm) is only close to the focallength f since the collimated LED is not a point source. The light beamis then refocused onto a collimation package assembled around anaspheric lens (f=10 mm, D=5 mm; NA˜D/2 f˜0.25, Ocean Optics Ltd) withinan anodized aluminum lens tube of length l=30 mm. Each LED is thuscoupled into one arm of a 2 m-long bifurcated silica core (Ø=600 μm,NA=0.22) optical fibre assembly (attenuation: 0.013 dB/m at 505nm-relative transmission: 82% (arm 1) and 87% (arm 2)).

Table 1 illustrates a power optimization of the system depicted in FIG.16. The power at 505 nm, P505, is read by the silicon photodiode whilethe distance between the LED collimator and the lens surface (L_(max)),the lens geometry, and the lens tube length (l) are changed. Only onearm of the bifurcated fibre is used.

TABLE 1 Lens I Lmax Psos Hemispherical 3 cm 20 mm 225.2 μW Hemispherical5 cm 18 mm 200.4 μW Hemispherical 8 cm 19 mm 222.8 μW Cylindrical 3 cm 9 mm 170.9 μW Cylindrical 5 cm  9 mm 164.1 μW Plano-convex 3 cm 16 mm220.9 μW Plano-convex 5 cm 15 mm 204.1 μW Plano-convex 8 cm 15 mm 173.7μW None None 12 mm 187.4 μW

For the bias values described above, when both arms of the fibre areused, the power at 505 nm read by the photodiode is 820 μW.

FIG. 17 is a plot of LED spectrum, light reflected, and residual LEDlight over a range of wavelengths (nm). FIG. 17 illustrates an LEDspectrum obtained in the cooled CCD (diodes: Ug=2.0 V; I=0.3 A; T=15°C.), calculated light reflected by the dichroic mirror, and residual LEDlight after the emitter. The insert shows the transmission curves of thedichroic and emitter. The plot indicates that there is a loss of powerwhen the incident light is reflected onto the sample. Additionally,light is red-shifted by 20 nm, which causes some of the LED light tointerfere with the carboxyfluorescein dyes. The choice of availableemitters and dichroic mirrors is limited by the dyes chosen to label themigrating DNA strands.

FIG. 18 is a plot of power of the LED-module over time. During a CEexperiment, it is crucial to reduce the fluctuations of the power of thelight source within less than 1%. FIG. 18 shows an example of the powerrecorded by the silicon photodiode (Probe S130A, Thorlabs) using theinternal calibration function to record the power emitted by thefiber-LED assembly at 505 nm over time. The diodes are supplied with a3.4 V DC voltage corresponding to a current of 1.4 A while the junctionis maintained at 15±1° C. The room is maintained at a temperature of 22°C. (R.H.=24%). The plot illustrates a temporal power evolution of theLED-module. The lines mark regimes where the power drops, e.g., by 4.8nW/s, 11.6 nW/s, and 5.0 nW/s. Overall, the power drops by about 1.95 μWover 5 min, i.e. 0.48%.

FIG. 19 is an illustration showing beam shape and size after the sampleobjective as measured by the laser camera. The asymmetry observed is dueto imperfections occurring when the two fibre arms are fused because ofthe large core diameter of the fibre, mismatches between the LED-to-LEDand the fiber-to-fibre distances, and tilt in the optical elements. Inthe results reported in the next section, the situation corresponding tothe single-spot will be used. One method includes adjusting all theoptics to obtain the maximum power at the merged end of the bifurcatedfibre. This can yield a misshapen light beam as the core size of eacharm is large (multimode fibre). To characterize the beam shape and sizeafter the microscope objective, i.e. at the entrance of the microchip, aCoherent Lasercam II'½ camera was placed on an {x,y,z} translation stageequipped with micrometer precision positioners and equipped with a LeicaHCX PL FLUOTAR (40X, NA=0.75, WD=0.40 mm) and adjustable filters. Theobjective was brought within ˜8 mm of the Olympus LUCPLFLN (20X,NA=0.45, WD=6.6-7.8 mm) mounted on the CE setup. This allowed directlyimaging the beam coming out of the fiber-LED assembly via the CE setup.The micrometer positioners allowed measuring the dimension of the beamwith a precision of 10 μm by moving the camera from one spot of theobtained beam profile image to another and reporting the traveleddistance. The power can be maximized by adjusting each opticalcollimation element (P=1.6 mW at 505 nm) (A) or the collimation elementscan be adjusted to give one single spot (P=1.0 mW at 505 nm) (B).

The system was employed for both static and dynamic fluorescencemeasurements. For the static fluorescence measurements, a 1 μMfluorescein, 6-FAM or rhodamine B solution is loaded into themicrochannel by using a standard laboratory vacuum line (13 PSI (0.88atm) depression) to pull the solution through the channel via2-mm-diameter access holes. The glass microchannel is anisotropicallyetched with fluorhydric acid (HF) in Schott Borofloat® low-fluorescenceglass (CE chip X8050, Micronit, B.V., The Netherlands). It issemi-elliptic with a width of 50 μm, a depth of 20 μm and a length of 85mm. The plastic microchannels are hot-embossed into a 1.1-mm-thickcyclic olefin copolymer (COC) sheet at ˜160° C. from a reactive-ionetched Si(100) master. The channel section is tapered with a 25° taperangle and has a width of 60 μm (top) and 39 μm (bottom), a depth of 20μand a length of 85 mm. Glass capillaries that are 1-cm-long (innerdiameter 4 mm) borosilicate are epoxy-glued onto the access holes to actas reservoirs (or wells). All solutions are filtered with a nylonmembrane (pore diameter: 0.2 μm) to remove small particles that willclog the channel.

The loaded chip is placed on the CE setup and the focus of the 63Xsample objective is aligned with the bottom of the channel. The emittedfluorescent light is gathered onto the 26.6 mm×6.7 mm (1024×255 pixels)array of the thermoelectrically cooled Andor CCD. The processed signalis vertically binned from the software-restricted central rowsirradiated by the light focused onto the spectrometer entrance slit. TheCCD is cooled down to −50° C. to reduce the binned dark counts to 270while the exposure time is 0.05 s.

FIGS. 21A and 21B are plots of CCD signal v/s wavelengths. The plotsindicate the vertically-binned signal from a 1 μM 6-FAM solution loadedinto a glass microchannel (A) and a 1 μM fluorescein solution loadedinto a plastic COC channel (B). The counts from the same microchannelfilled with water are subtracted to take into account theautofluorescence of the glass or plastic microdevice. The power emittedfrom the system is 0.98 mW and 1.03 mW at 505 nm for glass and COC,respectively. This is obtained by supplying the two LEDs (placed inseries) with a constant current of 0.74 A, which corresponds to avoltage of 7.0 V. Due to the choice of filters (emitter cut-on: T50 at535 nm), only the tail of the fluorophore emission is observed(fluorescein: 8.^(em) _(max)=513 at pH=13, 6-FAM: 8^(em) _(ma)x=517 atpH=9. The signal-to-noise ratio is 87 for 1 μM 6-FAM in glass and 36 for1 μM fluorescein in COC. The SNR is lower in glass because 6-FAM isknown to photobleach faster than fluorescein. The detection limitparameters for glass and plastic CE microdevices are summarized in Table2.

TABLE 2 Device Power at Maximum signal-to- material Fluorophore 505 nmcounts noise ratio Glass 1 uM 6-FAM 0.98 mW 720 36 COC 1 uM fluorescein1.03 mW 1750 87For dynamic fluorescence measurements, glass microchannels are loadedwith reagents similar to the reagents for the static measurementtesting, but a first sequence of reagents are flushed through themicrodevice to reduce the effect of the electroosmotic flow (EOF) thatopposes the electrophoretic flow and results in peak distortion from aGaussian shape and therefore loss of resolution. EOF arises from there-equilibration of the electrical double layer arising from the surfacecharge of the microchannel walls after the perturbation caused by themigrating charges under the electric field. The EOF can be efficientlycontrolled by using a coating polymer matrix such-aspoly-N-hydroxyethylacrylamide (pHEA) dissolved in water at 0.1% w/v.

The DNA fragments are separated by electrophoretically migrating withina sieving polymer matrix such as POP-5™ (Applied Biosystems, Inc.), amixture of polyacrylamides in an appropriate buffer, according to theirsize and interactions with the polymer network. After the pHEA coatinghas been applied, IX A.C.E.™ buffer (Amresco, Inc.) is flushed into thechannel by vacuum followed by POP-5™. A 1 μM solution of a poly-adenineoligonucleotide labeled with 6-FAM is placed in the sample well and willbe electrokinetically injected in the separation channel via across-injection geometry. 1X A.C.E.™ buffer is placed in the samplewaste, buffer waste, and waste wells to ensure ionic conductivity in thewhole device.

FIG. 21 is a plot of CCD signal v/s time for dynamic fluorescencemeasurements. The plot indicates fully binned CCD signal showing thepeak corresponding to the elution of the 1 μM oligonucleotide (elutiontime, t_(el)=77 s) detected by the optical module. The nature of thepeak is confirmed by the spectrum obtained in the CCD at t=77 s. It issimilar to the peak shown in FIG. 20A. The signal-to-noise ratio of 10can be improved by uniformly heating the chip to 50° C. The plot showsthe result of the migration of the oligonucleotide while the LED-fibreassembly delivers about 980 μW at 505 nm. The two LEDs, placed inparallel, are supplied with 3.9 V (I=1.9 A) while the junction is keptat 15° C. The migration field in the separation channel is 110 V/cm.

In this manner, an optical excitation module capable of visualizing a 1μM oligonucleotide migrating in a glass microchannel loaded with asieving matrix is assembled and tested. The output fibre beam size anddivergence, the power distribution in the beam exiting the fibreassembly as well as the output power stability over time approach thespecifications of existing LIF setups. A modified epifluorescencemicroscope arrangement is used in conjunction with a lightweight compactfixed spectrograph built around ion-etched grating and aligned with acooled Charge-Coupled Device (CCD) camera for added sensitivity.Fluorescent dyes such as fluorescein, 6-carboxyfluorescein (6-FAM) andrhodamine B can be detected in conventional plastic (cyclic olefincopolymer) and glass microchannels at submicromolar levels. A migratingsingle-stranded oligonucleotide DNA fragment (10-mer) labeled with 6-FAMcan also be detected with high signal-to-noise ratio whenelectrophoretically migrated in the microchannels at 100 V/cm. LEDsoperated in conjunction with Peltier elements controlled by aProportional Integrative Derivative (PID) module can be used to replacebulky, expensive and power-consuming Argon ion lasers conventionallyused in Laser Induced Fluorescence (LIF) Capillary Electrophoresis (CE)experiments. The LEDs in the system can be HP803-CN obtained fromRoithner LaserTechnik GmBH or Luxeon Star series from Philips LumiledLighting Company that offer LEDs emitting at 505±15 nm with a full-widthat half maximum of 20 nm. The LEDs are available with a Lambertianprofile with a half-cone angle of 75°, which is not suited for microchipapplications. However, these are high power LEDs with a nominalradiometric output power of 45 or 80 mW. When properly collimated, theavailable power becomes relevant to applications of DNA detection by CE.

While this specification contains many specifics, these should not beconstrued as limitations on the scope of the disclosure or of what maybe claimed, but rather as descriptions of features specific toparticular implementations of the disclosure. Certain features that aredescribed in this specification in the context of separateimplementations can also be implemented in combination in a singleimplementation. Conversely, various features that are described in thecontext of a single implementation can also be implemented in multipleimplementations separately or in any suitable subcombination. Moreover,although features may be described above as acting in certaincombinations and even initially claimed as such, one or more featuresfrom a claimed combination can in some cases be excised from thecombination, and the claimed combination may be directed to asubcombination or variation of a subcombination.

Similarly, while operations are depicted in the drawings in a particularorder, this should not be understood as requiring that such operationsbe performed in the particular order shown or in sequential order, orthat all illustrated operations be performed, to achieve desirableresults. In certain circumstances, multitasking and parallel processingmay be advantageous. Moreover, the separation of various systemcomponents in the implementations described above should not beunderstood as requiring such separation in all implementations, and itshould be understood that the described program components and systemscan generally be integrated together in a single software product orpackaged into multiple software products.

Thus, particular implementations of the disclosure have been described.Other implementations are within the scope of the following claims. Forexample, the actions recited in the claims can be performed in adifferent order and still achieve desirable results. In someimplementations, the sharpness of the cut-on edge of the dichroic mirrorcan be improved and the lower wavelength T₅₀ can be shifted to a lowerwavelength to improve the signal-to-noise ratio. In someimplementations, the diodes can be operated in a pulsed AC mode wherethe “on” time is synchronized with the frame acquisition of the CCDcamera, thereby also extending the lifetime of the LEDs. In someimplementation, a customized LED array can be used that does not havethe mold that yields divergent light. In some implementations, thecollimation parts can be embedded in a rigid casing made, e.g., fromblack anodized aluminum.

In some implementations, the LED-based detection system described inthis disclosure can be used as the microfluidic electrophoresis systemthat is described in the attachment, which is enclosed as part of thepresent disclosure.

5) Size Standards

The size standards used in the invention are beneficially stored withinthe formamide pump liquid.

The size standards may be provided according to the form detailed inInternational Patent Application no PCT/GB2009/002186, the contents ofwhich are incorporated herein by reference, particularly with respect tothe provision of and use of size standards which operate within a singleCE channel, together with the sample being considered.

Instrument Performance

The result of the above embodiment is the provision of an instrument,cartridge and operating method which provides quick, reliable sampleanalysis, whilst doing so at a wide variety of locations and whenoperated by a wide variety of people.

By way of abilities are performance, the invention provides a fullyintegrated instrument capable of performing extraction, PCR,electrophoresis and analysis, whilst requiring minimal training and/orintervention by the user. In its optimum form, a fully automated systemfrom start to finish is provided, the user simply needing to load thecartridge into the instrument and start it.

The modular nature of the instrument allows for upgrading of one or moremodules without impact on the other modules. The data output format hasbeen carefully selected to allow the analysis of the data outputted by avariety of existing analysis software applications, such is I³ ofForensic Science Service Limited, and future software applications.

The end result of the analysis may be a profile for the sample and/or anindication of a match between the sample and a database recorded sampleand/or other interpretation based data.

The use of a single cartridge type to handle a wide variety of samplefrom a wide variety of sources is beneficial. The methodology is able tohandle samples originating from buccal swabs, cotton and other softswabs, aqueous samples, clothing samples, cigarette butts, chewing gumand the like.

The methodology is also able to separate the useful DNA from residualcellular material, PCR inhibitors (such as ethanol, indigo etc) andchemical inhibitors.

The instrument is fully portable and so can be used in a wide variety oflocations. The fully sealed and protected nature of the cartridge meansthat contamination is not a risk, even where the instrument is usedoutside of laboratory standard conditions. The instrument operates off astandard mains power supply, 110-240V, 50 Hz, using a conventionalelectric plug.

With respect to the overall time, from the sample receiving step 202, tothe transmission away from the instrument in the data communication step210, the embodiment described provides this process in a time period of141 minutes. That time period can be reduced, including by the optionsand variables set out in the following paragraphs.

With respect to the sample receiving step 2002, the embodiment describedprovides this step in a time period of 2 minutes. Time periods ofbetween 20 seconds and 5 minutes are easily achievable, depending uponthe loading methodology used and the number of reagents or samples thatneed to be loaded.

With respect to the sample preparation step 202, the embodimentdescribed provides this step in a time period of 24 minutes. That timeperiod can be reduced by shortening the residence in one or more of thechambers, for instance the incubation chamber 358, and/or by reducingthe time separation between a valve being activated and reliance on theoutcome of the activation and/or by reducing the washing and/or elutionvolumes used. Time periods of between 15 to 30 minutes are easilyachievable.

With respect to the sample amplification step 204, the embodimentdescribed provides this step in a time period of 80 minutes. That timeperiod can be reduced by shortening the number of cycles used, theduration of one or more parts of a cycle and the time period afterintroduction to the chamber and before PCR starts and/or after PCRfinishes and before the sample is removed to the next stage. Again, thetime separation between a valve being activated and reliance on theoutcome of the activation is of significance. Time periods of between 60to 120 minutes are easily achievable.

With respect to the electrophoresis step 206, the embodiment describedprovides this step in a time period of 15 minutes. That time period canbe reduced by the use of higher voltages and/or faster migration mediain the capillary and/or reductions in the sample introduction time. Timeperiods of between 1 to 60 minutes are easily achievable. Thisfunctionality is achieved in an instrument weighing less than 10 kg andoccupying a footprint of less than 0.1 m².

Instrument Fields of Use

The structures and method discussed above are useful in theconsideration of a wide variety of samples, over and above forensicsamples. For instance, they can be used: the consideration of markertargets, diagnostic assays, disease markers, biobanking applications,STR based targets in transplants, identification of drug resistantmicroorganisms, blood testing, mutation detection, DNA sequencing andthe like. Food analysis, pharmogenetics and pharmogenomics are alsoareas of use. A wide variety of uses in the medical and/or biotech fieldcan make use of the invention.

The invention is also applicable in situations where familialrelationships need to be determined from DNA, for instance paternitytesting. Pedigree testing in animals is a further example.

The use of the invention in border control, security, customs situationsand other governmental type uses is beneficial.

1. A method of providing a storable sample, the method including: a)introducing a sample to a device; b) conveying at least a part of thesample to a receiving location to provide a storable sample, wherein astorable sample of a sample on which one or more processes and/orreactions are performed is provided.
 2. A method according to claim 1 inwhich the receiving location is provided in a section of the device thatis detachable from the device
 3. A method according to claim 2 in whichthe section is detached from the device by breaking the material joiningthe section and the device.
 4. A method according to claim 1 in whichthe storable sample is provided before one or more processes and/orreactions are performed.
 5. A method according to claim 1 in which thestorable sample is provided after one or more processed and/or reactionsare performed.
 6. A method according to claim 5 in which the one or moreprocesses and/or one or more reactions include one or more of: celllysis, mixing, a surface based reaction, washing, elution, selectiveseparation of DNA from one or more other materials, application of amagnetic field, removal of a magnetic field, amplification, PCR,detection and denaturation.
 7. A method according to claim 1 in whichthe method provides a storable sample which is one part of the sampleand one or more other parts of the sample are used in one or moreprocesses and/or reactions.
 8. A method according to claim 1 in whichthe method includes one or more of the following steps: passing thesample through one or more channels and/or chambers to mix the samplewith one or more fluids and/or solids; increasing the temperature of thesample and/or a mixture including the sample, preferably whilst in achamber; holding the sample and/or a mixture including the sample in achamber for a period of time; passing the sample through one or morefurther channels and/or further chambers; retaining at least a part ofthe sample in a chamber, preferably on a surface of one or more solids,preferably using a magnetic field; washing at least another part of thesample from the chamber where the at least a part of the sample isretained; eluting the retained part of the sample into a fluid.
 9. Amethod according to claim 1 in which the method includes transferring atleast a part of the sample from a reaction chamber to the receivinglocation.
 10. A method according to claim 1 in which the method providessample to a reaction chamber, with part of the sample progressing to thereceiving location when the amount of sample in the reaction chamberexceeds a predetermined amount.
 11. A method according to claim 1 inwhich the method includes sealing the channel leading to the receivinglocation.
 12. A method according to claim 1 in which the method includessealing the channel on the device side of the location where the channelis detached when the section is detached from the device.
 13. A device,the device having: a) an entry location; b) a channel connected to theentry location; c) a receiving location, the receiving location beingconnected to the channel.
 14. A device according to claim 13, whereinthe receiving location is detachable from the device.
 15. A deviceaccording to claim 13 in which the receiving location is provided in asection of the device and an area or line of weakness is providedbetween the device and the section.
 16. A device according to claim 13in which the device is provided with a first valve in the section forsealing the channel between the receiving location and the part of thechannel disrupted when the section is detached from the device.
 17. Adevice according to claim 13 in which the device is provided with asecond valve not on the section for sealing the channel between the partof the channel disrupted when the section is detached from the deviceand the remainder of the device.
 18. A device according to claim 13 inwhich the receiving location has an inlet in the top of the receivinglocation, the device having an orientation of use, and the receivinglocation has an outlet in the top of the receiving location.
 19. Adevice according to claim 13 in which the section is provided with anidentifier.
 20. A device according to claim 19 in which the identifierhas the same identifier information or includes the same identifierinformation as an identifier provided on the remainder of the device.21. A device according to claim 13 in which the device provides one ormore processing locations and/or reaction locations between the entrylocation and the receiving location.
 22. A device according to claim 21in which the one or more processing locations are channels and/orchambers and the processing locations include one or more of: a mixinglocation, a washing location, a selective separation location for DNAfrom one or more other material, an amplification process location, alocation at which a magnetic field is applied and/or removed and/orvaried, a cell lysis location, a surface based reaction location.
 23. Adevice according to claim 13 in which the device includes a reactionchamber connected to the receiving location, the reaction chamber beinga PCR reaction chamber.
 24. A device in which the device includes one ormore chambers, a chamber being provided with an inlet channel whichleads to an inlet, an outlet channel which leads away from an outlet anda by-pass channel, wherein the fluid flow switches from the inletchannel to the by-pass channel when a predetermined volume of fluid isprovided in the chamber.
 25. A device according to claim 24 in which theresistance to fluid flow provided by the outlet and/or outlet channel isgreater than the resistance to fluid flow provided by the inlet and/orinlet channel.
 26. A device according to claim 24 in which theresistance to fluid flow provided by the outlet and/or outlet channel isgreater than the resistance to fluid flow provided by the by-passchannel.
 27. A device according to claim 24 in which the path of leastresistance for the fluid is through the inlet and into the chamber untilthe fluid reaches the outlet and/or outlet channel.
 28. A deviceaccording to claim 24 in which the path of least resistance for thefluid is through the by-pass channel once the fluid has reached theoutlet and/or outlet channel.
 29. A device according to claim 24 inwhich the chamber includes a support location for one or more particles.30. A device according to claim 29 in which the one or more particlesprovide one or more or all the reagents for a reaction.
 31. A deviceaccording to claim 30 in which the reaction is an amplification.
 32. Adevice according to claim 24 in which the support location define aposition of rest for the one or more particles, with the position ofrest providing that the one or more particles do not block or obscure aninlet to and/or outlet from the chamber.
 33. A device according to claim24 in which the inlet for a sample is provided in the upper 20% of theheight of the chamber.
 34. A device according to claim 24 in which theoutlet for the sample is provided in the upper 20% of the height of thechamber.
 35. A device according to claim 24 in which the by pass channelconnects a part of the inlet channel to a part of the outlet channel.36. A device according to claim 24 in which one or more dimensions ofthe outlet channel are smaller than the corresponding dimension of theinlet channel.
 37. A device according to claim 24 in which thecross-sectional area of the outlet and/or outlet channel is smaller thanthe cross-sectional area of the inlet and/or inlet channel.
 38. A deviceaccording to claim 24 in which the inlet channel and the outlet channelto the chamber are sealable by one or more valves.
 39. A deviceaccording to claim 24 in which a channel leading to a chamber isprovided with a sealable location and a channel leading from thatchamber is provided with a further sealable location, wherein at one ormore points in time, heat is applied to 90% or more of the volume of thechannels and chamber between the location and the further location. 40.A device according to claim 39 in which the heat is applied by one ormore heating elements in contact with a surface facing the channels andchambers.
 41. A device according to claim 40 in which the heatingelements are in contact with a a surface facing at least 90% of thevolume of the channels and chamber between the location and furtherlocation.
 42. A device according to claim 24 in which the chamber isprovided with an inlet channel for a displacement fluid and an outletchannel leading to one or more further chambers, the inlet channel forthe displacement fluid being the inlet, the outlet channel for thedisplacement fluid being the outlet.
 43. A device according to claim 24in which the by-pass channel is provided with a valve and the closing ofthe valve provides that the resistance to fluid flow provided by theoutlet and/or outlet channel is less than the resistance to fluid flowprovided by the by-pass channel.
 44. A device according to claim 24 inwhich the resistance to fluid flow provided by the outlet and/or outletchannel is greater than the resistance to fluid flow provided by theby-pass channel.
 45. A method of controlling the passage of one or morematerials within a device, the method including: moving one or morematerials from a channel into a chamber connected to the channel; movingone or more of the materials from the chamber into a channel connectedto the chamber, wherein the chamber is provided with an inlet channelwhich leads to an inlet, an outlet channel which leads away from anoutlet and a by-pass channel, wherein the fluid flow switches from theinlet channel to the by-pass channel when a predetermined volume offluid is provided in the chamber.
 46. A method according to claim 45 inwhich the chamber is provided with an inlet channel which leads to aninlet, an outlet channel which leads away from an outlet and a by-passchannel, wherein the fluid flow switches from the inlet channel to theby-pass channel when a predetermined volume of fluid is provided in thechamber.
 47. A method according to claim 45 in which the resistance tofluid flow provided by the outlet and/or outlet channel is greater thanthe resistance to fluid flow provided by the inlet and/or inlet channel.48. A method according to claim 45 in which the resistance to fluid flowprovided by the outlet and/or outlet channel is greater than theresistance to fluid flow provided by the by-pass channel.
 49. A methodaccording to claim 45 in which the path of least resistance for thefluid is through the inlet and into the chamber until the fluid reachesthe outlet and/or outlet channel.
 50. A method according to claim 45 inwhich the path of least resistance for the fluid is through the by-passchannel once the fluid has reached the outlet and/or outlet channel.